Eradicates Cancer Stem Cells
I was simply astounded by a publication appearing last week in Oncotarget on anti-cancer synergy of Doxycyline and vitamin C.(1) This is a really huge breakthrough in cancer research, in our quest for effective non-toxic cancer treatment. Working with MCF7 Breast cancer cell cultures, Lisanti’s group showed the combined use of Doxycycline and Vitamin C was a “lethal metabolic strategy for eradicating cancer stem cells”.(1)
Doxycline is a safe common antibiotic used for 50 years. I have seen patients coming into the office on doxycycline for months or even years for treatment of acne or rosacea. Likewise Vitamin C is about as safe as a substance as you can get. Clinical trial safety study on relapsed B-Cell Lymphoma patients receiving 75 grams of Vitamin C Intravenously reported no adverse effects.(47) Left image doxycycline courtesy of National Library of Medicine
Cancer Stem Cells Escape from Doxycycline Become Purely Glycolytic Phenotype
In an elegant study, Lisanti’s group created Doxycycline resistant cancer stem cells by successive passage of the cells through higher doses of Doxycycline treatments. Most of the cells were killed by the Doxycycline. However the few surviving cells were then allowed to multiply and repopulate, and were again treated with higher doses of doxyxycline. This process was repeated until final cells were indeed Doxycyline resistant, they were immune to the antibiotic.
Above left image Figure 12 from Lisanti (1)
The Dox-Resistant cancer cells were now sensitive to eradication with metabolic perturbation from high dose vitamin C. Vitamin C acts as a glycolysis inhibitor, by targeting (GAPDH) Glyceraldehyde 3-phosphate dehydrogenase, 6th step in glycolysis. Vitamin C also and depletes the (NAD) nicotinamide adenine dinucleotide pool. High dose IV vitamin C easily reached serum concentrations for these lethal effects in the clinical setting.
A few other drugs and natural substances were also effective, namely Berberine, Chloroquin, Atovaquone (45-46), Niclosamide etc. ) The authors state,
“understanding the metabolic basis of Doxycycline-resistance has ultimately helped us to develop a new synthetic lethal strategy, for more effectively targeting Cancer Stem Cells (CSCs). ”
Left Image figure 10 from Lisanti(1) Fig A effect of 2DG (2-de-oxyglucose) on both plain MC7 breast cancer stem cells and Dox-resistant cells. Fig B: effect of Vitamin C on Dox Resistant breast cancer stem cells. 100% (complete) cell death at 500 micromolar=0.5 millimolar.
Metformin and Cancer Stem Cells
Lisanti’s group found Berberine effective against cancer stem cells. Metformin is another agent with similarities to berberine which targets cancer stem cells by inhibiting Complex One in the mitochondrial electron transport chain. Working with a pancreatic cancer cell model, Dr Patricia Sanchez in Cell Metabolism 2015 found that Metformin was effective as a cancer stem cell agent. (44) Dr Sanchez found that cancer stem cells rely on OXPHOS (oxidative Phosphorylation) while non-cancer stem cells were highly glycolytic. (44) Metformin treatment killed most of the cancer stem cells. However resistant stem cell clones emerged with a glycolytic phenotype. As noted above, this conversion to a glycolytic phenotype is exactly what Lisanti’s group found when treating breast cancer stem cells with Doxycyline. This glycolytic phenotype was then vulnerable to lethal effects of high dose IV vitamin C serving as glycolysis inhibitor.
Dr Sanchez was disappointed to find that pancreatic tumors developed resistance to Metformin progressed in a more aggressive form in cancer xenografts. On the other hand, treatment with Menadione (Vit K3), which both inhibited complex one and increased ROS, was lethal to the cancer xenografts without inducing resistant cell types.
Salinomycin 3BP Bogata Colombia
Salinomycin, another promising cancer stem cell agent, was used in an anecdotal case report from Bogata Colombia by Jason Williams MD and Marc Rosenberg MD. Twenty One rounds of IV Salinomycin and 3-bromopyruvate was given on alternate days to a patient with small cell lung cancer. Follow up CAT scan showed disappearance of the patient’s lung mass. Although results were encouraging, further Salinomycin trials in 20 or so additional patients provided only temporary remission from cancer, with later emergence of resistant cell types.(personal communication Jason Williams MD) Following these disappointing results, research efforts were re-directed towards other modalities such as image guided ablation and immunotherapy. Perhaps results would have been better with the use of other combination drugs such as Doxycycline, High Dose IV Vitamin C, Metformin, Atovaquone, Artesunate, Alpha Lipoic Acid etc.
This highlights the dangers of Cancer Stem Cell treatments which merely induce resistant cancer cell types which are more aggressive and more difficult to treat. Another problem is knowing when the treatment has eradicated all the cancer stem cells. At the present time we don’t have a good method for determine this, and so we don’t know when to stop treatment. If treatment is stopped too soon, then resistant cancer stem cells are left behind to induce a relapse. This is a major problem which awaits further research. This will be discussed in part two.
Dr Lisanti’s group found that Doxycyline combination therapy for eradication of cancer stem cells worked with other drugs such as the OXPHOS inhibitor, Atovaquone, an anti-malaria drug which inhibits complex III of the mitochondrial electron transport chain.(1)(46) Atavaquone’s chemical structure is similar to CoQ-10, and serves to block activity of Co-Q10 thereby inhibiting respiration in the cancer stem cell. Atavaquone is already FDA approved for prevention of Pneumocyctis pneumonia in immunosuppressed patients, and easily reaches effective serum levels with routine dosage of 750 mg BID with food.(45-46)
Ignoring the Cancer Stem Cells – the Failure of Oncology
For rapidly proliferating cancer cell types chemotherapy will provide a temporary remission, or reduction in tumor size. However, cancer stem cells are unaffected by chemotherapy and will induce cancer relapse. The more aggressive, highly proliferative cell types relapse within months while the more indolent cell types take longer, and relapse after a few years. Clearly, targeting cancer stem cells is imperative in order to prevent cancer relapse after treatment. Unfortunately, current day oncology has failed the cancer patient by ignoring cancer stem cells, and blindly forging ahead with the old chemotherapy protocols, as if medical science is still in the 1960’s, and nothing has changed.
Exemestane – Aromatase Inhibitor for Breast Cancer
Regardless of serum estrogen level which may be quite low in post-menopausal women, many tumors use intracrine estrogen production to stimulate cell growth. The tumor cells contain upregulated aromatase to produce estrogen locally. In this scenario, aromatese blocker such as exemestane is beneficial even when serum estrogen is quite low.
Exemestane is a third generation irreversible aromatase inhibitor, and a conventional oncology success story that stands out.(52-53) In addition to its aromatase blocking activity, exemestane metabolites cause very effective mitochondrial mediated apoptosis of breast cancer cells.(52) Exemestane also seems effective in lung cancer which frequently expresses estrogen receptors and has aromatase activity (53)(64). Exemestane may synergize with Doxycycline in treatment of mesothelioma (63)(16).
In-Situ Aromatase Activity in Skin Cancers
Similar to breast cancer cells, skin cells have aromatase activity upregulated in skin cancers to stimulate growth via intratumoral, in-situ, estrogen production (intracrine) independent of serum concentrations which may be quite low in post-menopausal women. Blocking aromatase activity has been shown effective for skin cancers (65-68) After all, breast tissue is an appendage of the skin, so estrogen production by the skin via aromatase activity is not unexpected.(65-68)
In- Situ (Intracrine) Aromatase Activity in Colon Cancer
One does not usually associate estrogen production with colonic epithelium or colon cancer cells. However, Dr Sato in 2012 found this to be the case.(74) He states:
“All these results demonstrate that colon carcinoma expresses functional aromatase, and that estrogens are locally synthesized in the tumor tissues.”(74)
Synergy of MTOR Inhibition with Exemestane
A number of studies show synergistic cancer cell killing effect when an MTOR inhibitor drug is added to the aromatase inhibitor (exemestane).(69-72) Note: Itraconazole is a potent MTOR inhibitor.
A combination of exemestane, doxycycline, itraconazole, fenofibrate, clarithromycin etc might be suggested for the breast cancer patients already on high dose IV vitamin C. Other Aromatase producing cancer cell types might also benefit from such a combination of drugs which block molecular pathways in cancer cells.
Bone Metastesis on Zolendronic Acid
In those with bone metastasis from breast cancer already on Zoledronic Acid, addition of Doxcycline to the Zoledronic Acid might be synergistic and more effective.(54-55)
Exemestane and Anti-Cancer Metabolites
In the drug development for exemestane, researchers may have accidentally stumbled upon a highly effective anti-cancer drug by virtue of the metabolites of exemestane which seem to have a different biological effect inducing “cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation”.(61) Dr Cristina Amaral in 2015 says:
“Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation... It was also concluded that….the biological effects of (exemestane) metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites.”(61)
More on Doxycycline – Benefits in Ascites and Pleural Effusions
Back in the day when I worked as an interventional radiologist, we commonly removed malignant pleural fluid from breast cancer patients, and then injected doxycycline as a “sclerosing agent”. In 1994, Dr Wakai discovered that Doxycycline (TCN) suppresses malignant effusions by suppressing tumor growth.(60) He says:
“it appears that TCNs (Doxycycline) injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.”(60)
Doxycycline as Stand Alone Anti-Cancer Drug
Studies show that doxycycline may be considered effective stand alone anti-cancer therapy. Doxycycline inhibited a B-cell lymphoma cell line in vitro and in mouse xenograft models (5), Doxycycline has activity in colon cancer (9), T lymphoblastic leukemia (10) melanoma (11) prostate and breast cancer (12-15), mesothelioma (16) oral squamous cell (17), glioblastoma (18), leukemia (19), Jurkat T lymphocytes (20) Colorectal cancer cells (21) Needless to say, docycycline is more effective when used in combination with drugs that block additional molecular pathways. One of these drugs is the antibiotic, Clarithromycin (Azithromycin) which is synergistic with Doxycycline and Vitamin C as discussed in my previous article on Clarithromycin.
Conclusion: My hat comes off in admiration and thanks to Michael Lisanti and his group. This Doxycycline/ Vitamin C combination is a dramatic breakthrough in finding an effective targeted cancer stem cell eradication strategy. Hopefully, this technique will be incorporated and routinely used on the oncology wards. In the mean time, print out this article and give it to your oncologist. Ask for and demand the hospital provide Doxycycline and high dose Intravenous vitamin C for your family members undergoing chemotherapy for cancer.
Update 2018: Latest from Michael Lisanti Lab
Diphenyleneiodonium chloride (DPI), inhibitor of NADPH/NADH oxidase, and mitochondrial OXPHOS inhibitor was found to be anti-cancer stem cell agent at low concentrations by Michael Lisanti’s Lab.(50) Authors state:
“DPI induced a state of metabolic-quiescence, which potently prevented CSC (cancer stem cell) propagation, selectively depleting cancer stem cell population….DPI, at a concentration of only 10 nM, effectively inhibited OXPHOS and ultimately ATP production, by more than 90% overall. As a consequence, DPI treatment induced a reactive glycolytic phenotype, resulting in the production of high levels of L-lactate….Since DPI targets flavin-containing enzymes, its effects on mitochondrial function may be explained by the pharmacological induction of an acute Riboflavin (Vitamin B2) deficiency.”(50)
DPI is also being considered for anti-mycobacterial therapy, as an Anti-Tuberculosis antibiotic. (51)
This article is part one. For part two click here.
Articles with related interest:
Jeffrey Dach MD
7450 Griffin Road, Suite 190
Davie, Fl 33314
1) De Francesco, E. M., Michael Lisanti et al. “Vitamin C and Doxycycline: a synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs).” Oncotarget (2017). Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs).
2) Combining vitamin C with antibiotics destroys cancer stem cells
By Honor Whiteman published Tuesday 13 June 2017 670
3) Vitamin C and Antibiotic Combo Can Kill Cancer Cells
Posted on June 13, 2017, 6 a.m. in Cancer Immune System Vitamins
Researchers have shown that a combination of antibiotics and Vitamin C can destroy cancer stem cells before they promote the growth of fatal tumors.
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Doxycyline Effective for B Cell Lymphoma
5) Pulvino, Mary, et al. “Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline.”
In searching for small-molecule compounds that inhibit proliferation and survival of diffuse large B-cell lymphoma (DLBCL) cells and may, therefore, be exploited as potential therapeutic agents for this disease, we identified the commonly used and well-tolerated antibiotic doxycycline as a strong candidate. Here, we demonstrate that doxycycline inhibits the growth of DLBCL cells both in vitro and in mouse xenograft models. In addition, we show that doxycycline accumulates in DLBCL cells to high concentrations and affects multiple signaling pathways that are crucial for lymphomagenesis. Our data reveal the deneddylating activity of COP-9 signalosome (CSN) as a novel target of doxycycline and suggest that doxycycline may exert its effects in DLBCL cells in part through a CSN5-HSP90 pathway. Consistently, knockdown of CSN5 exhibited similar effects as doxycycline treatment on DLBCL cell survival and HSP90 chaperone function. In addition to DLBCL cells, doxycycline inhibited growth of several other types of non-Hodgkin lymphoma cells in vitro. Together, our results suggest that doxycycline may represent a promising therapeutic agent for DLBCL and other non-Hodgkin lymphomas subtypes.
6) Barbie, David A., and Brian K. Kennedy. “Doxycycline: new tricks for an old drug.” Oncotarget 6.23 (2015): 19336.
7) Peiris-Pagès, Maria, Federica Sotgia, and Michael P. Lisanti. “Doxycycline and therapeutic targeting of the DNA damage response in cancer cells: old drug, new purpose.” Oncoscience 2.8 (2015): 696.
8) free pdf
Saikali, Zeina, and Gurmit Singh. “Doxycycline and other tetracyclines in the treatment of bone metastasis.” Anti-cancer drugs 14.10 (2003): 773-778.
9) Onoda, Toshinao, et al. “Tetracycline analogues (doxycycline and COL‐3) induce caspase‐dependent and‐independent apoptosis in human colon cancer cells.” International journal of cancer 118.5 (2006): 1309-1315.
10) Iwasaki, Hiromichi, et al. “Doxycycline induces apoptosis by way of caspase-3 activation with inhibition of matrix metalloproteinase in human T-lymphoblastic leukemia CCRF-CEM cells.” Journal of Laboratory and Clinical Medicine 140.6 (2002): 382-386.
11) Sun, Tao, et al. “Doxycycline inhibits the adhesion and migration of melanoma cells by inhibiting the expression and phosphorylation of focal adhesion kinase (FAK).” Cancer letters 285.2 (2009): 141-150.
12) Lokeshwar, Bal L. “Chemically modified non-antimicrobial tetracyclines are multifunctional drugs against advanced cancers.” Pharmacological research 63.2 (2011): 146-150.
13) Zhang, Le, et al. “Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer.” Cell Cycle just-accepted (2016): 00-00.
14) Tang, Xiaoyun, et al. “Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.” Molecular cancer 16.1 (2017): 36.
Doxycycline suppressed both LPA- and TNFα-induced nuclear translocation of NF-κB and blocked the LPA-induced secretion of IL-6, CCL2 and CXCL2 in cancer cells. TNFα-induced nuclear NF-κB transcriptional activity was also inhibited by doxycycline. Under basal condition without stimulation, doxycycline was able to decrease the transcriptional activity of nuclear NF-κB by ~50%
The equivalent dose for human is ~4 mg/kg/day calculated by equivalent surface area dosage conversion factor, which is 240 mg/day for 60 kg of body weight. The typical dose of doxycycline is 100–200 mg/day and the maximum dose is 300 mg/day for more serious infections, such as syphilis.
15) Fife, Rose S., and George W. Sledge Jr. “Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells.” The Journal of laboratory and clinical medicine 125.3 (1995): 407-411.
16) Rubins, Jeffrey B., et al. “Inhibition of mesothelioma cell growth in vitro by doxycycline.” Journal of Laboratory and Clinical Medicine 138.2 (2001): 101-106.
17) Shen, Ling-Chang, et al. “Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma–in vitro and in vivo studies.” Oral oncology 46.3 (2010): 178-184.
18) Wang-Gillam, Andrea, et al. “Anti-tumor effect of doxycycline on glioblastoma cells.” Journal of Cancer Molecules 3.5 (2007): 147-153.Anti-Tumor Effect of Doxycycline on Glioblastoma Cells
19) Tolomeo, Manlio, et al. “Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines.” British journal of pharmacology 133.2 (2001): 306-314.
20) Liu, Jian, Charles A. Kuszynski, and B. Timothy Baxter. “Doxycycline induces Fas/Fas ligand-mediated apoptosis in Jurkat T lymphocytes.” Biochemical and biophysical research communications 260.2 (1999): 562-567.
21) Onoda, Toshinao, et al. “Doxycycline inhibits cell proliferation and invasive potential: combination therapy with cyclooxygenase-2 inhibitor in human colorectal cancer cells.” Journal of Laboratory and Clinical Medicine 143.4 (2004): 207-216.
22) Fife, Rose S., et al. “Effects of tetracyclines on angiogenesis in vitro.” Cancer letters 153.1 (2000): 75-78.
23) Sagar, Jayesh, et al. “Doxycycline in mitochondrial mediated pathway of apoptosis: a systematic review.” Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents) 10.7 (2010): 556-563.
24) Richards, Christopher, Liron Pantanowitz, and Bruce J. Dezube. “Antimicrobial and non-antimicrobial tetracyclines in human cancer trials.” Pharmacological research 63.2 (2011): 151-156.
25) van den Bogert, Coby, et al. “Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis.” Cancer research 46.7 (1986): 3283-3289.
26) Kroon, Albert M., et al. “The mitochondrial genetic system as a target for chemotherapy: tetracyclines as cytostatics.” Cancer letters 25.1 (1984): 33-40.
27) van den Bogert, Coby, Bert HJ Dontje, and Albert M. Kroon. “The antitumour effect of doxycycline on a T-cell leukaemia in the rat.” Leukemia research 9.5 (1985): 617-623.
Antibiotics Eradicate Cancer Stem Cells
28) Lamb, Rebecca, et al. “Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease.” Lamb Rebecca Antibiotics that target mitochondria effectively eradicate cancer stem cells 2015 OncoTarget Lamb Rebecca Antibiotics that target mitochondria effectively eradicate cancer stem cells 2015 OncoTarget
Finally, recent clinical trials with doxycycline and azithromycin (intended to target cancer-associated infections, but not cancer cells) have already shown positive therapeutic effects in cancer patients, although their ability to eradicate cancer stem cells was not yet appreciated.
Doxycycline for Lymphoma
29) Ann Hematol. 2015 Apr;94(4):575-81. Long-term outcomes of first-line treatment with doxycycline in patients with previously untreated ocular adnexal marginal zone B cell lymphoma. . Han JJ1, Kim TM, Jeon YK, Kim MK, Khwarg SI, Kim CW, Kim IH, Heo DS.
Author information 1Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, South Korea.
Ocular adnexal lymphoma (OAL) has been associated with Chlamydophila psittaci infection, for which doxycycline has been suggested as a treatment option. We conducted this study to evaluate the long-term results of first-line doxycycline treatment in patients with OAL. Ninety patients withhistologically confirmed OAL with marginal zone B cell lymphoma were enrolled. Each patient received one or two cycles of doxycycline (100 mg bid) for 3 weeks. After a median follow-up period of 40.5 months (8-85), the 5-year progression-free survival (PFS) rate was 60.9 %. All patients were alive at the last follow-up date. Thirty-one patients (34 %) showed local treatment failure without systemic spread. However, PFS rate in these patients was 100 % after salvage chemotherapy and/or radiotherapy.
PFS was independently predicted in multivariate analysis by the tumor-node-metastasis (TNM) staging (hazard ratio [HR], 4.35; 95 % confidence interval [CI], 2.03-9.32; P < 0.001) and number of cycles of doxycycline (HR, 0.31; 95 % CI, 0.14-0.69; P = 0.004). No serious adverse event was reported during doxycycline therapy. In conclusion, first-line doxycycline therapy was effective and safe.
Patients who failed to respond to doxycycline therapy were successfully salvaged with chemotherapy and/or radiotherapy without compromising long-term outcomes. Patients with T1N0M0 disease could be considered good candidates for first-line doxycycline.
13 patients antibiotics alone for gastric lymphoma – HP eradication regimen
30) Ann Hematol. 2015 Jun;94(6):969-73. doi: 10.1007/s00277-014-2298-3. Epub 2015 Jan 13. Antibiotic treatment as sole management of Helicobacter pylori-negative gastric MALT lymphoma: a single center experience with prolonged follow-up. Raderer M1, Wöhrer S, Kiesewetter B, Dolak W, Lagler H, Wotherspoon A, Muellauer L, Chott A.
Relatively little is known about the long-term outcome of patients with Helicobacter pylori (HP)-negative gastric lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) with antibiotic therapy as sole management. We have analyzed all patients with HP-negative gastric MALT lymphoma undergoing antibiotic therapy as sole management of their disease. HP negativity was defined as negative histology, breath test and serology, and response to treatment, survival and long-term outcome was assessed together with clinico-pathological characteristics including t(11; 18) (q21; q21) translocation. Out of 97 patients with gastric MALT lymphoma, 24 were HP-negative, and 13 (5 females and 8 males) underwent only antibiotic management for initial therapy. Eight had stage I and five were found to have stage II disease, with three patients suffering from an underlying autoimmune disease. Antibiotic therapy consisted of standard HP eradication regimens consisting of clarithromycin in all patients, along with metronidazole in seven and amoxicillin in six plus a proton-pump inhibitor. After a median follow-up of 95 months (42-, 181+), 12/13 patients are alive. Six patients with stage I disease achieved an objective response (five complete (CR) and one partial remission, 46 %), four had stable disease (lasting 11-27 months), and three progressed. All patients with stable disease received chemotherapy, but only one patient due to clear cut progression. One patient relapsed 23 months after initial CR, and achieved a second CR with antibiotics now lasting 87 months. These results indicate that a relevant percentage of patients with HP-negative gastric MALT lymphoma may benefit from antibiotic therapy and do not require additional oncological therapies. Our data suggest that the remissions seen in these patients might be durable as evidenced by prolonged follow-up in our series.
31) Kiesewetter, Barbara, and Markus Raderer. “Antibiotic therapy in nongastrointestinal MALT lymphoma: a review of the literature” Blood 122.8 (2013): 1350-1357.
A single course of oral doxycycline at a dose of 100 mg given twice a day for 3 weeks was the most popular regimen and was used by most investigators.14⇓⇓-17,19⇓⇓-22 By contrast, Kim and coworkers19 added a second course after an interval of 3 weeks for patients with residual eye-related symptoms after the initial cycle. The activity of a 6-month oral application of 500 mg clarithromycin twice a day was assessed in an Italian pilot study,18 assuming potential additional direct anticancer effects of macrolide antibiotics through changes in apoptotic mechanisms of tumor cells. In addition, 1 patient received HP eradication as first-line treatment of OAML. CR was achieved in 23 patients (18%) out of the collective of all 131 patients reported. Thirty-six (27%) had a PR
32) Ferreri, Andrés JM, et al. “Bacteria-eradicating therapy with doxycycline in ocular adnexal MALT lymphoma: a multicenter prospective trial.” Journal of the National Cancer Institute 98.19 (2006):1375-1382.
Background: An association between ocular adnexal MALT lymphoma (OAL) and Chlamydia psittaci (Cp) infection has been proposed, and recent reports suggest that doxycycline treatment causes tumor regression in patients with Cp-related OAL. The effectiveness of doxycycline treatment in Cp-negative OAL has not been tested. Methods: In a prospective trial, 27 OAL patients (15 newly diagnosed and 12 having experienced relapse) were given a 3-week course of doxycycline therapy. Objective lymphoma response was assessed by computerized tomography scans or magnetic resonance imaging at 1, 3, and 6 months after the conclusion of therapy and every 6 months during follow-up. Cp infection in patients was determined by touchdown enzyme time-release polymerase chain reaction (TETR-PCR). Statistical tests were two-sided. Results: Eleven patients were Cp DNA–positive and 16 were Cp DNA negative. Doxycycline was well tolerated. At a median follow-up of 14 months, lymphoma regression was complete in six patients, and a partial response (≥50% reduction of all measurable lesions) was observed in seven patients (overall response rate [complete and partial responses] = 48%). Lymphoma regression was observed in both Cp DNA–positive patients (seven of 11 experienced regression) and Cp DNA–negative patients (six of 16 experienced regression) (64% versus 38%; P = .25, Fisher’s exact test). The three patients with regional lymphadenopathies and three of the five patients with bilateral disease achieved objective response. In relapsed patients, response was observed both in previously irradiated and nonirradiated patients. The 2-year failure-free survival rate among the doxycycline- treated patients was 66% (95% confidence interval = 54 to 78), and 20 of the 27 patients were progression free. Conclusions: Doxycycline is a fast, safe, and active therapy for Cp DNA–positive OAL that was effective even in patients with multiple failures involving previously irradiated areas or regional lymphadenopathies. The responses observed in PCR-negative OAL may suggest a need for development of more sensitive methods for Cp detection and investigation of the potential role of other doxycycline-sensitive bacteria.
Ferreri et al conducted a prospective phase 2 clinical trial of 27 patients (15 newly diagnosed and 12 relapsed) with OAML, using doxycycline 100 mg orally twice daily for 3 weeks. Partial or complete lymphoma regression after antibiotic therapy was observed in 7 of 11 Cp-positive and 6 of 16 Cp-negative patients, with an overall response rate of 48%. The 2-year failure-free survival rate among patients treated with doxycycline was 66%
Abramson et al84 treated 3 patients with biopsy-proven conjunctival MALT lymphoma with antibiotic therapy, resulting in 2 complete remissions and 1 partial response.
Husain et al43 conducted a meta-analysis, identifying 4 studies with a total of 42 patients who had been treated with oral doxycycline.
full free pdf
33) Husain, Amina, et al. “Meta–analyses of the association between Chlamydia psittaci and ocular adnexal lymphoma and the response of ocular adnexal lymphoma to antibiotics.” Cancer 110.4 (2007): 809-815.
34) Abramson DH, Rollins I, Coleman M. Periocular mucosa-associated lymphoid/low grade lymphomas: treatment with antibiotics. Am J Ophthalmol. 2005;140:729–730. Am J Ophthalmol. 2005 Oct;140(4):729-30.
To report on the treatment of primary mucosa-associated lymphoid tumors (MALT)/low grade lymphomas of the conjunctiva/orbit treated solely with systemic antibiotics. DESIGN: Retrospective interventional case series.
METHODS: Three adult patients with biopsy/marker proven MALT lymphomas of the conjunctiva/orbit were treated with systemic antibiotics and followed for signs of local or systemic relapse.
RESULTS: All three patients showed a response to antibiotics based on clinical, ultrasonographic, and MRI/CT imaging studies. Two patients have had complete remissions (42 months follow-up) and one a partial remission (18 months). No systemic relapses have occurred.
CONCLUSION: MALT/low grade lymphomas of the conjunctiva/orbit respond to systemic antibiotic therapy and may have complete remissions.
35) Chatzispyrou, Iliana A., et al. “Tetracycline antibiotics impair mitochondrial function and its experimental use confounds research.” Cancer research 75.21 (2015): 4446-4449.
36) Moullan, Norman, et al. “Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research.” Cell reports 10.10 (2015): 1681-1691.
37) Oncotarget. 2016 Nov 15; 7(46): 75954–75967.
Doxycycline is an NF-κB inhibitor that induces apoptotic cell death in malignant T-cells
Carolina V. Alexander-Savino,1 Matthew S. Hayden,1 Christopher Richardson,2 Jiyong Zhao,3 and Brian Poligone1,4
Doxycycline is an inexpensive and widely used tetracycline, commonly known for its antibiotic properties, that was first synthesized from chlortetracycline in 1967 . Chlortetracycline is the parent structure for all tetracyclines and is naturally found in Streptomyces aureofaciens . Doxycycline’s antibiotic effects come from its ability to bind to the bacterial ribosome’s 30s subunit and inhibit protein synthesis.
38) Toberer F, Hartschuh W, Hadaschik E. Primary cutaneous CD4+ small- to medium-sized pleomorphic T-cell lymphoma: temporary remission by oral doxycycline. JAMA dermatology. 2013;149:956–959.
Preventing Metastases With Anti-platelet drugs and Anti-Progesterone Drug
39) Aspirin, lysine, mifepristone and doxycycline combined can effectively and safely prevent and treat cancer metastasis: prevent seeds from gemmating on soil.
Wan L, Dong H, Xu H, Ma J, Zhu Y, Lu Y, Wang J, Zhang T, Li T, Xie J, Xu B, Xie F, Gao Y, Shao J, Tu X, Jia L Oncotarget. 2015 Nov 3; 6(34):35157-72.
We then demonstrated that metapristone (the active mifepristone metabolite) has a safe and effective profile as a cancer metastasis chemopreventive agent by inhibiting adhesion of CTCs to vascular endothelium
Besides, mifepristone significantly decreased expression of focal adhesion kinase that is related to cell spreading and survival. Interestingly, it seems like that there are some similarity between embryo implantation and tumor metastasis , which constructs the base for the abortifacient mifepristone to act as a metastatic chemopreventive. Patients already took mifepristone for as long as 14 years . The safety profile of mifepristone makes it well-suited for a safe metastatic chemopreventive candidate.
40) Med Res Rev. 2014 Sep;34(5):979-1000. doi: 10.1002/med.21311. Epub 2014 Mar 1.
The unique pharmacological characteristics of mifepristone (RU486): from terminating pregnancy to preventing cancer metastasis.
Chen J1, Wang J, Shao J, Gao Y, Xu J, Yu S, Liu Z, Jia L.
Mifepristone (RU486) is a born-for-woman molecule discovered three decades ago. Unlike those antihypertensive and antipsychotic pharmaceutical blockbusters, this abortifacient offers relatively low profit potential. Current understanding of mechanism of action of mifepristone and its on-going clinical trials are changing our views on the drug beyond its abortifacient scope. Here we briefly review its metabolism and pharmacokinetic properties including its unique enterohepatic circulation, its mechanisms of actions involving antiprogesterone and antiglucocorticoid, growth inhibition of various cancer cell lines, suppression of invasive and metastatic cancer potential, downregulation of Cdk2, Bcl-2, and NF-kappa B, interference of heterotypic cell adhesion to basement membrane, and cell migration.
We comprehensively analyze recent results from preclinical and clinical studies using mifepristone as an anticancer drug for breast, meningioma, and gliomas tumors in the central nervous system, prostate cancer, ovarian and endometrial cancer, and gastric adenocarcinoma. Although mifepristone has more benefits for global public health than we originally thought, its effect as a postmetastatic chemotherapeutic agent is limited. Nonetheless, owing to its unique safe, metabolism and other pharmacological properties, metapristone (the primary metabolite of mifepristone) may have potential for cancer metastatic chemoprevention.
mifepristone 200 mg daily x 2 years
41) Ji, Yongli, et al. “Double-blind phase III randomized trial of the antiprogestin agent mifepristone in the treatment of unresectable meningioma: SWOG S9005.” Journal of Clinical Oncology 33.34 (2015): 4093-4098.
Patients were randomly assigned to either oral mifepristone 200 mg daily or placebo for 2 years (Fig 1). The dose of 200 mg was chosen for its antiprogesterone activity and its minimal antiglucocorticoid activity.22 Patients carried a warning card to alert medical personnel that the investigational treatment could cause subclinical adrenal insufficiency and to recommend administration of exogenous glucocorticoids in case of emergency.
42) Ozsvári, Béla, Rebecca Lamb, and Michael P. Lisanti. “Repurposing of FDA-approved drugs against cancer–focus on metastasis.” Aging (Albany NY) 8.4 (2016): 567.
43) Starving Cancer Cells Out of Existence. Doctors Knock Out Cancer Stem Cells With A Common Antibiotic and Finish Off Treatment-Resistant Stem Cells With Vitamin C
By Bill Sardi June 12, 2017 Lew Rockwell
44) Patricia Sancho, et al. “MYC/PGC-1a Balance Determines the Metabolic Phenotype and Plasticity of Pancreatic Cancer Stem Cells.” Cell Metabolism 22 (2015): 1-16. MYC Determines Metabolic Phenotype Pancreatic Cancer Stem Cells Metformin Patricia Sancho Cell Metabolism 2015
The anti-diabetic drug metformin targets pancreatic cancer stem cells (CSCs), but not their differentiated progenies (non-CSCs), which may be related to distinct metabolic phenotypes. Here we conclusively demonstrate that while non-CSCs were highly glycolytic, CSCs were dependent on oxidative metabolism (OXPHOS) with very limited metabolic plasticity. Thus, mitochondrial inhibition, e.g., by metformin, translated into energy crisis and apoptosis. However, resistant CSC clones eventually emerged during treatment with metformin due to their intermediate glycolytic/respiratory phenotype. Mechanistically, suppression of MYC and subsequent increase of PGC-1α were identified as key determinants for the OXPHOS dependency of CSCs, which was abolished in resistant CSC clones. Intriguingly, no resistance was observed for the mitochondrial ROS inducer menadione and resistance could also be prevented/reversed for metformin by genetic/pharmacological inhibition of MYC. Thus, the specific metabolic features of pancreatic CSCs are amendable to therapeutic intervention and could provide the basis for developing more effective therapies to combat this lethal cancer.
Methods of treating cancer with atovaquone-related compounds WO 2015050844 A1
Described herein are, inter alia, methods for decreasing the growth of a cancer cell, the method comprising delivering to a target cancer cell a growth-inhibitory amount of an atovaquone-related compound, wherein, prior to the delivery, an increased level of activation of the mTOR pathway in the cancer compared to a control level of activation of the mTOR pathway has been found. Also provided are methods for determining the susceptibility of cancer to treatment with an atovaquone-related compound and for assessing the success of therapy with such a compound.
46) Oncotarget. 2016 Jun 7;7(23):34084-99. doi: 10.18632/oncotarget.9122.
Repurposing atovaquone: targeting mitochondrial complex III and OXPHOS to eradicate cancer stem cells. Fiorillo M1,2,3, Lamb R1, Tanowitz HB4, Mutti L5, Krstic-Demonacos M5, Cappello AR3, Martinez-Outschoorn UE6, Sotgia F1,2, Lisanti MP1,2.
Atovaquone is an FDA-approved anti-malarial drug, which first became clinically available in the year 2000. Currently, its main usage is for the treatment of pneumocystis pneumonia (PCP) and/or toxoplasmosis in immune-compromised patients. Atovaquone is a hydroxy-1,4-naphthoquinone analogue of ubiquinone, also known as Co-enzyme Q10 (CoQ10). It is a well-tolerated drug that does not cause myelo-suppression. Mechanistically, it is thought to act as a potent and selective OXPHOS inhibitor, by targeting the CoQ10-dependence of mitochondrial complex III. Here, we show for the first time that atovaquone also has anti-cancer activity, directed against Cancer Stem-like Cells (CSCs). More specifically, we demonstrate that atovaquone treatment of MCF7 breast cancer cells inhibits oxygen-consumption and metabolically induces aerobic glycolysis (the Warburg effect), as well as oxidative stress. Remarkably, atovaquone potently inhibits the propagation of MCF7-derived CSCs, with an IC-50 of 1 μM, as measured using the mammosphere assay. Atovaquone also maintains this selectivity and potency in mixed populations of CSCs and non-CSCs. Importantly, these results indicate that glycolysis itself is not sufficient to maintain the proliferation of CSCs, which is instead strictly dependent on mitochondrial function. In addition to targeting the proliferation of CSCs, atovaquone also induces apoptosis in both CD44+/CD24low/- CSC and ALDH+ CSC populations, during exposure to anchorage-independent conditions for 12 hours. However, it has no effect on oxygen consumption in normal human fibroblasts and, in this cellular context, behaves as an anti-inflammatory, consistent with the fact that it is well-tolerated in patients treated for infections. Future studies in xenograft models and human clinical trials may be warranted, as the IC-50 of atovaquone’s action on CSCs (1 μM) is >50 times less than its average serum concentration in humans.
Atovaquone can be administered alone as a liquid suspension (brand name Mepron) or in combination with Proguanil (brand name Malarone). Atovaquone is a highly lipophilic compound, with limited solubility in water. The bioavailability of atovaquone is dependent on its formulation and the diet, and its absorption is enhanced by high-fat food intake. Importantly, with current oral formulations, the average serum concentration of atovaquone in humans is > 50 μM.
Atovaquone is a quinone that functions as a competitive inhibitor of co-enzyme Q10
When atovaquone suspension was administered in humans with food at the standard regimen of 750 mg twice daily, the average steady-state plasma concentration was 21.0 ± 4.9 μg/mL, and the minimum plasma concentration was 16 ± 3.8 μg/mL . It should be noted that in our experiments we have effectively ablated mammosphere formation with 10 μM atovaquone, which corresponds to 3.66 μg/mL. Thus, the clinically relevant, therapeutic plasma concentration of atovaquone is 5-times higher than the concentration that completely blocks the expansion of CSCs.
47) KAWADA, Hiroshi, et al. “Phase I Clinical Trial of Intravenous L-ascorbic Acid Following Salvage Chemotherapy for Relapsed B-cell non-Hodgkin’s Lymphoma.” The Tokai journal of experimental and clinical medicine 39.3 (2014): 111-115. Intravenous Ascorbic Acid Following Chemotherapy for Relapsed B-Cell Lymphoma KAWADA Hiroshi Tokai journal 2014
48) Wang, S. Q., et al. “New application of an old drug: Antitumor activity and mechanisms of doxycycline in small cell lung cancer.” International journal of oncology 48.4 (2016): 1353.
49) Cancer stem cells, doxycycline and glioblastoma
Jacob Schor ND, FABNO September 6, 2017
50) Ozsvari, Bela, et al. “Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy.” Aging (Albany NY) 9.12 (2017): 2610. Michael Lisanti Group
we conducted a high-throughput drug screen to discover novel mitochondrial inhibitors. More specifically, this screening assay was engineered to select chemical entities that potently reduce ATP levels, but do not induce cell death. With this strategy, DPI was identified a top hit compound. More specifically, DPI induced a state of metabolic-quiescence, which potently prevented CSC propagation (IC-50 = 3.2 nM). Complementary findings were also provided using well-established CSC markers (CD44 and CD24). Surprisingly, DPI treatment selectively depleted the CSC sub-population (CD44+/CD24-) from the total cancer cell population.
DPI, at a concentration of only 10 nM, effectively inhibited OXPHOS and ultimately ATP production, by more than 90% overall. As a consequence, DPI treatment induced a reactive glycolytic phenotype, resulting in the production of high levels of L-lactate.
Since DPI targets flavin-containing enzymes, its effects on mitochondrial function may be explained by the pharmacological induction of an acute Riboflavin (Vitamin B2) deficiency. Riboflavin-deficiency directly targets the steady-state expression levels of OXPHOS-related proteins of mitochondrial complex I and II, as predicted
51) J Antimicrob Chemother. 2017 Nov 1;72(11):3117-3121. doi: 10.1093/jac/dkx277.
Biological evaluation of diphenyleneiodonium chloride (DPIC) as a potential drug candidate for treatment of non-tuberculous mycobacterial infections.
Singh AK1, Thakare R1, Karaulia P1, Das S1, Soni I1, Pandey M2, Pandey AK2, Chopra S1, Dasgupta A1.
Novel drug discovery against non-tuberculous mycobacteria is beset with a large number of challenges including the existence of myriad innate drug resistance mechanisms as well as a lack of suitable animal models, which hinders effective translation. In order to identify molecules acting via novel mechanisms of action, we screened the Library of Pharmacologically Active Compounds against non-tuberculous mycobacteria to identify such compounds.
Methods:Whole-cell growth inhibition assays were used to screen and identify novel inhibitors. The hit compounds were tested for cytotoxicity against Vero cells to determine the selectivity index, and time-kill kinetics were determined against Mycobacterium fortuitum. The compound’s ability to synergize with amikacin, ceftriaxone, ceftazidime and meropenem was determined using fractional inhibitory concentration indexes followed by its ability to decimate mycobacterial infections ex vivo. Finally, the in vivo potential was determined in a neutropenic murine model mimicking mycobacterial infection.
Results: We have identified diphenyleneiodonium chloride (DPIC), an NADPH/NADH oxidase inhibitor, as possessing potent antimicrobial activity against non-tuberculous mycobacteria. DPIC exhibited concentration-dependent bactericidal activity against M. fortuitum and synergized with amikacin, ceftriaxone, ceftazidime and meropenem. When tested in a murine neutropenic M. fortuitum infection model, DPIC caused a significant reduction in bacterial load in kidney and spleen. The reduction in bacterial count is comparable to amikacin at a 100-fold lower concentration.
Conclusions:DPIC exhibits all properties to be repositioned as a novel anti-mycobacterial therapy and possesses a potentially new mechanism of action. Thus, it can be projected as a potential new therapeutic against ever-increasing non-tuberculous mycobacterial infections.
52) Amaral, Cristina, et al. “Apoptosis and autophagy in breast cancer cells following exemestane treatment.” PLoS One 7.8 (2012): e42398.
Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G0/G1 or in G2/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.
53) Koutras, Angelos, et al. “Antiproliferative effect of exemestane in lung cancer cells.” Molecular cancer 8.1 (2009): 109.
Combine Doxycycline and Zoledronic Acid for Breast Cancer Bone Metastasis 2007
54) Duivenvoorden, W. C. M., et al. “Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer.” British journal of cancer 96.10 (2007): 1526.
Bone is one of the most frequent sites for metastasis in breast cancer patients often resulting in significant clinical morbidity and mortality. Bisphosphonates are currently the standard of care for breast cancer patients with bone metastasis. We have shown previously that doxycycline, a member of the tetracycline family of antibiotics, reduces total tumour burden in an experimental bone metastasis mouse model of human breast cancer. In this study, we combined doxycycline treatment together with zoledronic acid, the most potent bisphosphonate. Drug administration started 3 days before the injection of the MDA-MB-231 cells. When mice were administered zoledronic acid alone, the total tumour burden decreased by 43% compared to placebo treatment. Administration of a combination of zoledronic acid and doxycycline resulted in a 74% decrease in total tumour burden compared to untreated mice. In doxycycline- and zoledronate-treated mice bone formation was significantly enhanced as determined by increased numbers of osteoblasts, osteoid surface and volume, whereas a decrease in bone resorption was also observed. Doxycycline greatly reduced tumour burden and could also compensate for the increased bone resorption. The addition of zoledronate to the regimen further decreased tumour burden, caused an extensive decrease in bone-associated soft tissue tumour burden (93%), and sustained the bone volume, which could result in a smaller fracture risk. Treatment with zoledronic acid in combination with doxycycline may be very beneficial for breast cancer patients at risk for osteolytic bone metastasis.
Doxycylcine in Treatment of Bone Metastasis 2003
55) Anticancer Drugs. 2003 Nov;14(10):773-8.
Doxycycline and other tetracyclines in the treatment of bone metastasis.
Saikali Z1, Singh G.
The tetracycline family includes tetracycline, doxycycline and minocycline, all of which have been used as antibiotics effectively for decades. New uses emerged for these compounds after their effect on mitochondrial function was discovered. Cytostatic and cytotoxic activity of these compounds was shown against cell lines of various tumor origins. In addition, tetracyclines and chemically modified tetracyclines inhibit the activity of several matrix metalloproteinases (MMPs). Given the importance of these enzymes in tumor cell invasion and metastatic ability, the potential use of tetracyclines in cancer therapy needed to be investigated. Col-3, a chemically modified tetracycline, is now the subject of clinical trials in cancer patients. However, the potential of tetracyclines in cancer therapy takes on an added dimension in the bone. MMPs have been shown to be important mediators of metastasis formation in the bone, contributing largely to the morbidity of breast cancer and prostate cancer patients. The natural osteotropism of tetracyclines would allow them to be highly effective in the inhibition of MMPs produced by osteoclasts or tumor cells in the bone. This hypothesis has now been confirmed by experimental evidence showing that doxycycline reduces tumor burden in a mouse model of breast cancer-derived osteolytic bone metastasis. This effect is likely due to a combination of multiple roles of doxycycline, including MMP inhibition and a negative effect on osteoclast differentiation and survival. These encouraging results have now paved the way for an ongoing trial of doxycycline in early combination therapy for breast cancer and prostate cancer patients.
56) Zhang, Le, et al. “Doxycycline inhibits the cancer stem cell phenotype and epithelial-to-mesenchymal transition in breast cancer.” Cell Cycle 16.8 (2017): 737-745.
Experimental evidence suggest that breast tumors originate from breast cancer stem cells (BCSCs), and that mitochondrial biogenesis is essential for the anchorage-independent clonal expansion and survival of CSCs, thus rendering mitochondria a significant target for novel treatment approaches. One of the recognized side effects of the FDA-approved drug, doxycycline is the inhibition of mitochondrial biogenesis. Here we investigate the mechanism by which doxycycline exerts its inhibitory effects on the properties of breast cancer cells and BCSCs, such as mammosphere forming efficiency, invasion, migration, apoptosis, the expression of stem cell markers and epithelial-to-mesenchymal transition (EMT) related markers of breast cancer cells. In addition, we explored whether autophagy plays a role in the inhibitory effect of doxycycline on breast cancer cells. We find that doxycyline can inhibit the viability and proliferation of breast cancer cells and BCSCs, decrease mammosphere forming efficiency, migration and invasion, and EMT of breast cancer cells. Expression of stem cell factors Oct4, Sox2, Nanog and CD44 were also significantly downregulated after doxycycline treatment. Moreover, doxycycline could down-regulate the expression of the autophagy marker LC-3BI and LC-3BII, suggesting that inhibiting autophagy may be responsible in part for the observed effects on proliferation, EMT and stem cell markers. The potent inhibition of EMT and cancer stem-like characteristics in breast cancer cells by doxycycline treatment suggests that this drug can be repurposed as an anti-cancer drug in the treatment of breast cancer patients in the clinic.
57) Tang, Xiaoyun, et al. “Doxycycline attenuates breast cancer related inflammation by decreasing plasma lysophosphatidate concentrations and inhibiting NF-κB activation.” Molecular cancer 16.1 (2017): 36.
We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces.
In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting.
Results Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1β, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80+ macrophages and CD31+ blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate.
These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.
58) Barbie, David A., and Brian K. Kennedy. “Doxycycline: new tricks for an old drug.” Oncotarget 6.23 (2015): 19336.
Since their original discovery in 1948, tetracycline antibiotics have had a major impact on human health and molecular biology . Beyond their efficacy against a variety of infectious diseases, insights into resistance and discovery of the tetracycline repressor system has yielded an invaluable tool for the inducible control of gene expression. Though originally characterized as selective inhibitors of the bacterial 30S ribosomal subunit , tetracyclines appear also to have alternate molecular targets in human tissues and are effective in treating certain skin disorders such as acne and rosacea, which extends beyond a direct antimicrobial effect . Three separate recently published reports characterize “these off-target” activities in detail, suggesting that doxycycline might be repurposed as an anti-cancer therapeutic.
Pulvino et al. utilized the Connectivity Map  to search for compounds that reversed an NF-κB signature in HL60 cells, identifying multiple tetracycline family members, including doxycycline . Consistent with this observation, when they treated several different diffuse large B cell lymphoma (DLBCL) cell lines with doxycycline, the authors observed inhibition of NF-κB signaling coupled to decreased cell proliferation and survival. Doxycycline also perturbed STAT3 and ERK activation and reduced the levels of multiple different HSP90 client proteins in these cells, leading the authors to explore its effects on HSP90 activity. Interestingly, doxycycline treatment increased HSP90 ubiquitination and degradation, and more generally increased proteasome-dependent protein neddylation. Through a series of elegant studies, they then determined that these effects were mediated through direct inhibition of the JAMM family metalloproteinase CSN5. Notably, doxycycline was also effective at inhibiting the growth of DLBCL xenografts at physiologically achievable doses, which has prompted the translation of these findings into a clinical trial of single agent doxycycline in relapsed/refractory non-Hodgkins lymphoma patients.
Two other studies characterize a distinct but related role for doxycycline in inhibiting MCF7 breast cancer cell mammosphere formation [6, 7]. De Luca et al. determined that MCF7 mammospheres are particularly sensitive to perturbation of mitochondrial function . Consistent with this observation, over-expression of the transcription factor FOXM1, which promotes stem-like phenotypes, increased mammosphere formation together with upregulation of multiple mitochondrial proteins. Since recent work from this group also demonstrated that doxycycline could suppress tumor-sphere formation by targeting mitochondrial ribosomes, they tested the capacity of doxycycline to reverse FOXM1 driven spherogenesis. Doxycycline indeed prevented the capacity of FOXM1 to promote mammospheres, albeit at very high concentrations. Lamb et al. further determined that doxycycline treatment can inhibit mammosphere formation in primary breast cancer samples, and performed proteomics analysis of MCF7 cells to determine putative doxycycline targets. They noted marked downregulation of DNA-PK by doxycycline treatment, and determined that shRNA-mediated DNA-PK suppression or direct pharmacologic inhibition phenocopied doxycycline, disrupting MCF7 mammosphere formation. Since DNA-PK is involved in radio-resistance, they also evaluated whether doxycycline treatment could synergize with radiation to inhibit MCF7 mammospheres, and observed sensitivity of this subpopulation relative to cells in a monolayer. Finally and similar to Pulvino et al. , using a series of reporters, Lamb et al. found that doxycycline treatment of MCF7 cells impairs multiple other signaling pathways, including STAT3 and NRF1/2 .
Taken together, these studies suggest that doxycycline, a drug that has been utilized for over 50 years as an antimicrobial agent and more recently in dermatological conditions, could also have efficacy in certain cancers. Despite this promise, several barriers remain to effective translation of these findings to the clinic. First, while Pulvino et al. carefully determined that the doxycycline concentrations used in their experiments match clinical exposure , this was not fully explored in the breast cancer studies [5, 7]. Second, a discrete readout of effective target inhibition by doxycycline in human tumors will be necessary to understand whether the doses utilized in clinical trials are indeed high enough to yield prolonged suppression of the tumorigenic pathways. For example, despite equally strong evidence of antitumor efficacy of the antimalarial drug hydroxychloroquine in preclinical studies, treatment of pancreatic adenocarcinoma patients was ineffective and associated with inconsistent autophagy inhibition in peripheral blood cells . Perhaps most importantly, single agent therapy, especially in relapsed aggressive cancers, is less likely to be effective even with proven target inhibition. Thus, negative findings in the lymphoma study, for example, do not necessarily invalidate doxycycline as a useful agent, but may suggest the need to develop combinatorial approaches with more targeted BTK inhibitors, for example. Finally, the characterization of CSN5 and DNA-PK as putative doxycycline targets that mediate its anticancer activities supports the development of more potent and selective inhibitors of these enzymes, which, depending on their therapeutic window, could have an even greater impact for cancer patients.
J Lab Clin Med. 1995 Mar;125(3):407-11.
59) Effects of doxycycline on in vitro growth, migration, and gelatinase activity of breast carcinoma cells. Fife RS1, Sledge GW Jr.
Metastatic disease is one of the major causes of death from cancer in human beings. Several enzyme systems have been implicated in the metastatic process, but the metalloproteinases (MPs) appear to be the major group involved in most instances of neoplastic invasion. Increased MP activity has been correlated with the metastatic potential of many cancers, including breast cancer. MPs also play a role in tumor angiogenesis. Tetracyclines are antimicrobial agents that can suppress MP activity in a variety of tissues, including gingiva, bone, and cartilage. Several reports have indicated that tetracyclines can suppress tumor MPs as well. A synthetic tetracycline, doxycycline, inhibits migration of human MDA-MB-435 breast adenocarcinoma cells through a reconstituted basement membrane (Matrigel), an assay used as an in vitro surrogate for the in vivo process of tumor invasion through basement membranes. Additionally, doxycycline diminishes the proliferation of this breast cancer cell line and also decreases its gelatinolytic activity, as determined by gel zymography.
Ascites and Pleural Effusions
60) Mechanism of inhibitory actions of minocycline and doxycycline on ascitic fluid production induced by mouse fibrosarcoma cells.Wakai K1, Ohmura E, Satoh T, Murakami H, Isozaki O, Emoto N, Demura H, Shizume K, Tsushima T. Life Sci. 1994;54(11):703-9.
Semisynthetic tetracyclines (TCNs) are used for the management of malignant pleural effusions as sclerosing agents. However, their precise mechanism of actions are uncertain. In the present study, the mechanism of inhibitory effects of minocycline (MINO) and doxycycline (DOXY), on the accumulation of ascitic fluid induced by mouse fibrosarcoma (Meth-A) cells were investigated using male mice. Meth-A cells inoculated intraperitoneally elicited 2.5-4 ml of bloody ascites 10 days after implantation. The production of ascitic fluid was suppressed in a dose-related manner by daily intraperitoneal injections of MINO or DOXY, whereas vehicle (normal saline with 0.01N HCl) did not exert a significant effect. The inhibitory activity of these two substances was quite similar; one mg/mouse of MINO or DOXY inhibited the accumulation of fluid by 87% and 84%, respectively. The survival rate of Meth-A-bearing mice treated with MINO or DOXY was higher than that of the controls. Macroscopic examination of the peritoneal cavity did not reveal any obvious effects, such as adhesions, in mice treated with either MINO or DOXY. In vitro studies showed that MINO and DOXY suppressed Meth-A cell growth with IC50s of 5 microM and 8 microM, respectively. Maximal suppression (95%) was achieved at MINO and DOXY concentrations of 25 microM. The above observations suggest that MINO and DOXY inhibit the accumulation of ascites by a direct effect on Meth-A cell growth. Therefore, it appears that TCNs injected into the pleural cavity to manage malignant effusions in man exert their activity, at least in part, by suppressing malignant cell growth.
61) Amaral, Cristina, et al. “Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane.” The international journal of biochemistry & cell biology 69 (2015): 183-195.
Around 60-80% of all breast tumors are estrogen receptor-positive. One of the several therapeutic approaches used for this type of cancers is the use of aromatase inhibitors. Exemestane is a third-generation steroidal aromatase inhibitor that undergoes a complex and extensive metabolism, being catalytically converted into chemically active metabolites. Recently, our group showed that the major exemestane metabolites, 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione, as well as, the intermediary metabolite 6β-Spirooxiranandrosta-1,4-diene-3,17-dione, are potent aromatase inhibitors in breast cancer cells. In this work, in order to better understand the biological mechanisms of exemestane in breast cancer and the effectiveness of its metabolites, it was investigated their effects in sensitive and acquired-resistant estrogen receptor-positive breast cancer cells. Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation. Moreover, metabolites also induced autophagy as a promoter mechanism of apoptosis. In addition, it was demonstrated that metabolites can sensitize aromatase inhibitors-resistant cancer cells, by inducing apoptosis. Therefore, this study indicates that exemestane after metabolization originates active metabolites that suppress the growth of sensitive and resistant breast cancer cells. It was also concluded that, in both cell lines, the biological effects of metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites.
62) De Francesco, Ernestina Marianna, et al. “Targeting hypoxic cancer stem cells (CSCs) with Doxycycline: implications for optimizing anti-angiogenic therapy.” Oncotarget 8.34 (2017): 56126.
Nuvoli, Barbara, et al. “Exemestane blocks mesothelioma growth through downregulation of cAMP, pCREB and CD44 implicating new treatment option in patients affected by this disease.” Molecular cancer 13.1 (2014): 69.
64) free pdf Weinberg, Olga K., et al. “Aromatase inhibitors in human lung cancer therapy.” Cancer research 65.24 (2005): 11287-11291.
65) Pomari, Elena, et al. “Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.” The FASEB Journal 29.2 (2014): 508-524.
17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24–48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen.
66) Rev Endocr Metab Disord. 2016 Sep;17(3):247-258. Skin steroidogenesis in health and disease. Nikolakis G1, Stratakis CA2, Kanaki T3, Slominski A4, Zouboulis CC3.
The skin is an important extra-gonadal steroidogenic organ, capable of metabolizing various hormones from their precursors, as well as of synthesizing de novo a broad palette of sex steroids and glucocorticoids from cholesterol. In this manuscript, we review the major steroidogenic properties of human skin and we suggest steroidogenesis’ impairment as a cardinal factor for various pathological conditions such as acne, rosacea, atopic dermatitis, and androgenic alopecia.
67) Anticancer Drugs. 2002 Jun;13(5):521-31. Effects of tamoxifen on human squamous cell carcinoma lines of the head and neck. Hoffmann TK1, Bojar H, Eckel J, van Lierop A, Balz V, Friebe-Hoffmann U, Hauser U, Bier H.
Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.
68) Arch Oral Biol. 2006 Jul;51(7):612-20. Epub 2006 Feb 28. Aromatase expression in normal human oral keratinocytes and oral squamous cell carcinoma.Cheng YS1, Mues G, Wood D, Ding J.
Aromatase is the enzyme that catalyzes the conversion of androgen to oestrogen. Aromatase expression in extra-gonadal sites and local oestrogen synthesis play an important role in the physiological conditions and in the growth of certain neoplasms.The purpose of this study was to investigate aromatase expression in oral keratinocytes and oral squamous cell carcinoma (SCC).
DESIGN:Immunocytochemistry and RT-nested PCR were used to detect aromatase protein and mRNA expression in primary human oral epithelial cell culture and in an oral SCC cell line. Immunohistochemistry was used to detect aromatase protein expression in frozen and archival human tissue sections of normal oral epithelium and oral SCC.
RESULTS: Cytoplasmic immunostaining was found in normal oral keratinocytes and SCC cells in culture. The common coding region of aromatase mRNA was detected in the oral keratinocytes derived from five different normal individuals and in the SCC cell line. However, there were variations in aromatase exon 1 expression among normal oral keratinocyte samples. Cytoplasmic staining was found in normal oral epithelium and well-differentiated oral SCC but not in poorly differentiated oral SCC by immunohistochemistry.
CONCLUSION: Aromatase was expressed in normal oral keratinocytes and oral SCC both in cell culture and in tissues, indicating local oestrogen synthesis in normal and neoplastic conditions of oral epithelium.
Exemestane synergistic with MTOR inhibitor
69) Awada, Ahmad, et al. “The oral mTOR inhibitor RAD001 (everolimus) in combination with letrozole in patients with advanced breast cancer: results of a phase I study with pharmacokinetics.” European journal of cancer 44.1 (2008): 84-91.
70) Rudloff, Joëlle, et al. “The mTOR pathway in estrogen response: A potential for combining the rapamycin derivative RAD001 with the aromatase inhibitor Letrozole (Femara®) in breast carcinoma.” (2004): 1298-1298.
RAD001 is an mTOR pathway inhibitor in phase II clinical trials in oncology that exerts potent antiproliferative and antitumor activities. A number of breast carcinoma’s are dependent for proliferation on estrogens (E) synthesized from androgens by the aromatase enzyme. Letrozole (Femara®) is a potent and selective non-steroidal aromatase inhibitor used for the treatment of postmenopausal women with hormone-dependent breast cancers. We addressed the role of the mTOR pathway in the E-induced proliferative response of vector control (MCF7 3(1)) and aromatase-expressing (MCF7/Aro) breast cancer cell lines in vitro. Moreover, the effect of RAD001/Letrozole combinations on this response was assessed in MCF7/Aro cells. MCF7 3(1) and MCF7/Aro cells were sensitive to treatment with 20 nM RAD001; a significant G1 accumulation occurred (82 % vs 62 % and 81 % vs 62 % cells in G1 for MCF7 3(1) and MCF7/Aro, resp; p<0.05 χ2-test). RAD001 almost completely inhibited estradiol (E2)-induced proliferation in MCF7 3(1) cells and E2- and androstenedione (Δ4A)-induced proliferation in MCF7/Aro cells, suggesting that mTOR signaling is required for E2-induced proliferative response in MCF7 cells. To study the effect of RAD001/letrozole combinations on MCF7/Aro cell proliferation, sub-optimal and optimal concentrations were defined (10 and 100 nM letrozole; 0.2 and 2 nM RAD001, resp), which were combined or applied alone in E2-dependent proliferation assays. Each single agent displayed a concentration-dependent antiproliferative activity on Δ4A-driven cell proliferation (p<0.001; two-way ANOVA). However, when used in combination, inhibition of cell proliferation was increased. For example, in two experiments, the inhibitory effect of 100 nM letrozole (73 % and 41 % inhibition) and 0.2 nM RAD001 (52 % and 43 % inhibition) was increased to 87 % and 72 % when used in combination. Similarly, the inhibitory effect of 100 nM letrozole and 2 nM RAD001 (80 % and 78 % inhibition) was increased to 93 % and 89 % when used in combination. Statistical analyses indicated highly significant interactions between RAD001 and letrozole (p<0.001; two-way ANOVA), suggestive of strong combination effects. Moreover, further statistical analysis demonstrated that RAD001 and letrozole act in an additive/synergistic manner to inhibit MCF7/Aro cell proliferation. Importantly, no evidence of antagonism was observed. Based on the demonstrated importance of the mTOR pathway in E-induced proliferative response and the increased inhibitory potential of RAD001/letrozole combinations, combination of these agents hold promise for the treatment of estrogen-dependent breast cancers.
Note: Itraconazole is a potent MTOR inhibitor
72) Boulay, Anne, et al. “Dual inhibition of mTOR and estrogen receptor signaling in vitro induces cell death in models of breast cancer.” Clinical Cancer Research 11.14 (2005): 5319-5328.
73) Lee, Joycelyn JX, Kiley Loh, and Yoon-Sim Yap. “PI3K/Akt/mTOR inhibitors in breast cancer.” Cancer biology & medicine 12.4 (2015): 342.
74) Sato, Ryuichiro, et al. “Aromatase in colon carcinoma.” Anticancer research 32.8 (2012): 3069-3075.
Aromatase is one of the key estrogen-producing enzymes and is regarded as one of the therapeutic targets in estrogen receptor-positive breast cancer patients. Human colon carcinoma has also been recently proposed as being an estrogen-responsive malignancy, but the detailed status of aromatase has not yet been reported. Therefore, in this study, we evaluated the aromatase expression in colon carcinoma using immunohistochemistry and real-time polymerase chain reaction. Aromatase mRNA was significantly higher (p=0.03) in colon carcinoma than in the corresponding non-neoplastic mucosa (n=31). Aromatase immunoreactivity tended to be positively associated with the intratumoral concentration of estrogens (n=53), and in particular, the concentration of estradiol was significantly higher (p=0.02) in aromatase-positive cases in men. Aromatase immunoreactivity was detected in the cytoplasm of the carcinoma cells in 217/328 (65%) examined colon carcinoma cases. Aromatase immunoreactivity was significantly positively correlated with tubular differentiation, and inversely correlated with Ki-67 labeling index, although not necessarily correlated with the clinical outcome of the patients. All these results demonstrate that colon carcinoma expresses functional aromatase, and that estrogens are locally synthesized in the tumor tissues. The findings reported here could contribute to a better understanding of the actions of estrogen in colon carcinoma.
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