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Combination effect of doxorubicin and HIF inhibitor on MCF-7 CD44+/CD24- subpopulation cells in hypoxic condition

Abstract

Hypoxia-inducible factors (HIFs) and cancer stem cells (CSCs) are two challenging causes of radiotherapy and chemotherapy resistance, leading to most cases of failure and recurrence in breast cancer therapy. This study was conducted to investigated the inhibitory effect of combination therapy with doxorubicin (an anthracycline) and FM19G11 (an HIF inhibitor) on MCF-7 cells and their CSC-like cells (CSC-LCs). MCF-7 CSC-LCs with a CD44+/CD24- phenotype were sorted and characterized by flow cytometry. A combination of doxorubicin and FM19G11 caused more cytotoxic effects on MCF-7 and CSC-LCs compared to doxorubicin monotherapy. The largest synergistic effect was observed in CSC-LCs under hypoxic conditions; however, MCF-7 cells showed no synergism in normoxic conditions. The administration of doxorubicin and FM19G11 induced late apoptotic and necrotic cell death in MCF-7 and CSC-LCs. Additionally, G2 phase arrest was observed in both cells. Our results demonstrated that co-administration of FM19G11 and doxorubicin had a synergistic effect in hypoxia and improved drug resistance in breast cancer stem cells.

Keywords:
FM19G11; Combination Index; Hypoxia-induced factor; Cytotoxicity; Cancer stem cells

INTRODUCTION

Breast cancer has been recognized as the most prevalent cancer and the primary cause of cancer-related death among females (Torre et al., 2015Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87-108.). There are a variety of ameliorative therapies to improve the survival rate of breast cancer patients such as mastectomy, radiotherapy, hormone therapy, and chemotherapy (Theriault et al., 2013Theriault RL, Carlson RW, Allred C, Anderson BO, Burstein HJ, Edge SB, et al. Breast cancer, version 3.2013. J Natl Compr Canc Netw. 2013;11(7):753-61.). However, there is a pronounced therapeutic resistance to chemotherapy agents, endocrine therapy, and radiotherapy that has been attributed to the presence of breast cancer stem cells (BCSCs) (Economopoulou, Kaklamani, Siziopikou, 2012Economopoulou P, Kaklamani VG, Siziopikou K. The role of cancer stem cells in breast cancer initiation and progression: potential cancer stem cell-directed therapies. Oncologist. 2012;17(11):1394-401.). This phenomenon is mediated through multiple mechanisms such as overexpression of ABC transporters, particularly ABCG2 and P-glycoprotein efflux pumps, decreased reactive oxygen species, activation of DNA-damage checkpoints, and apoptosis resistance (Dean, Fojo, Bates, 2005Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer. 2005;5(4):275-84.; Phillips, McBride, Pajonk, 2006Phillips TM, McBride WH, Pajonk F. The response of CD24−/low/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst. 2006;98(24):1777-85.). CSCs, as a minute fraction of tumor cells, are characterized by self-renewal capability and anomalous activation of developmental signaling pathways. Genetic instability and epigenetic alterations contribute to the generation of CSCs from normal stem cells or differentiated cells, leading to hastened cancer establishment, metastasis, and recurrence. Thus, CSCs are prominent targets to surmount these obstacles (Nassar, Blanpain, 2016Nassar D, Blanpain C. Cancer stem cells: basic concepts and therapeutic implications. Annu Rev Pathol-Mech. 2016;11(1):47-76.; Zhao et al., 2018Zhao Y, Dong Q, Li J, Zhang K, Qin J, Zhao J, et al. Targeting cancer stem cells and their niche: perspectives for future therapeutic targets and strategies. Semin Cancer Biol . 2018;53:139-155.).

Another factor that increases cancer therapeutic resistance is the accumulation of hypoxia-induced factors (HIFs), which were first identified by Semenza et al. in (1991Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE. Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci U S A. 1991;88(13):5680-4.). Indeed, HIFs as transcriptional factors engage in cell survival, proliferation, telomerase activation, genetic instability, angiogenesis, stem cell-related networks, glucose metabolism, invasion, metastasis and immune evasion (Burroughs et al., 2013Burroughs SK, Kaluz S, Wang D, Wang K, Van Meir EG, Wang B. Hypoxia inducible factor pathway inhibitors as anticancer therapeutics. Future Med Chem. 2013;5(5):553-72.). Likewise, HIFs maintain self-renewal feature, a hallmark of CSCs, through modulation of pluripotency transcription factors (Oct-4, Nanog, Sox-2, and c-Myc) (Schöning, Monteiro, Gu, 2017Schöning JP, Monteiro M, Gu W. Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia-inducible factors HIF 1α and HIF 2α. Clin Exp Pharmacol Physiol. 2017;44(2):153-61.). To date, different inhibitors have been developed to inhibit the HIF-1α functions by blocking protein synthesis and stability, hetero-dimerization, HIF-1α/DNA binding, and transactivation (Bhattarai, Xu, Lee, 2017Bhattarai D, Xu X, Lee K. Hypoxia-inducible factor-1 (HIF-1) inhibitors from the last decade (2007 to 2016): A “structure-activity relationship” perspective. Med Res Rev. 2017;38(4):1404-42.), among which FM19G11 is a small molecule with low toxicity that inhibits HIFα protein at the mRNA level (Moreno-Manzano et al., 2010Moreno-Manzano V, Rodríguez-Jiménez FJ, Aceña-Bonilla JL, Fustero-Lardíes S, Erceg S, Dopazo J, et al. FM19G11, a new hypoxia-inducible factor (HIF) modulator, affects stem cell differentiation status. J Biol Chem. 2010;285(2):1333-42.). FM19G11 exerts its inhibitory effects via regulation of stemness maintenance genes, which is an ideal feature for spinal cord injuries and cancer therapy (Alastrue-Agudo et al., 2018Alastrue-Agudo A, Rodriguez-Jimenez FJ, Mocholi EL, De Giorgio F, Erceg S, Moreno-Manzano V. FM19G11 and ependymal progenitor/stem cell combinatory treatment enhances neuronal preservation and oligodendrogenesis after severe spinal cord injury. Int J Mol Sci. 2018;19(1):200.; Moreno-Manzano et al., 2010Moreno-Manzano V, Rodríguez-Jiménez FJ, Aceña-Bonilla JL, Fustero-Lardíes S, Erceg S, Dopazo J, et al. FM19G11, a new hypoxia-inducible factor (HIF) modulator, affects stem cell differentiation status. J Biol Chem. 2010;285(2):1333-42.). Moreover, it could efficiently increase endothelium-dependent vasodilatation via recruiting the PI3K/Akt/eNOS pathway (El Assar et al., 2015El Assar M, Sanchez-Puelles JM, Royo I, Lopez-Hernandez E, Sanchez-Ferrer A, Acena JL, et al. FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway, independently of mTOR/HIF-1α activation. Br J Pharmacol. 2015;172(5):1277-91.).

The objective of this study is to investigate the suppressive effect of doxorubicin and FM19G11 on proliferation and cell cycle distribution of MCF-7 cells and their CD44+/CD24- cancer stem cell-like cells (CSC-LCs) in normoxic and hypoxic conditions.

MATERIAL AND METHODS

Chemicals, reagents, and drugs

RPMI 1640 and Penicillin-Streptomycin Solution were obtained from Biosera (East Sussex, UK). MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide was purchased from Carl Roth (Karlsruhe, Germany). Annexin V-FITC and propidium iodide (PI) were provided from Invitrogen (CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Doxorubicin was purchased from Pfizer (New York, USA). FM19G11 ([2-oxo-2-(p-tolyl) ethyl] 3-[(2, 4-dinitrobenzoyl) amino] benzoate, 3-[(2, 4-Dinitrobenzoyl) amino]-benzoic acid 2-(4-methylphenyl)-2-oxoethyl ester) was obtained from Sigma Aldrich.

Cell culture

MCF-7, a human breast adenocarcinoma cancer cell line, was purchased from Pasteur Institute Cell Bank of Iran (Tehran, Iran). CD44+/CD24- cells (CSC-LCs) were sorted and characterized as previously reported (Sajadian et al., 2015Sajadian S, Vatankhah M, Majdzadeh M, Kouhsari SM, Ghahremani MH, Ostad SN. Cell cycle arrest and apoptogenic properties of opium alkaloids noscapine and papaverine on breast cancer stem cells. Toxicol Mech Methods. 2015;25(5):388-95.). The cells were maintained in 89% RPMI medium, 10% fetal bovine serum (FBS; Gibco, USA) and 1% Penicillin-Streptomycin at 37 ºC and 5% CO2. As for the hypoxic condition, the cells were grown in a hypoxia incubator chamber with 1% O2 at 37 ºC.

Viability assay

MTT assay was used to determine the cytotoxic effect of doxorubicin and FM19G11, alone and in combination, on the viability of CSC-LCs and MCF-7 cells. The cells were seeded at 1×104 per well in a 96-well plate and incubated at 37 ºC overnight. Afterward, the cells were exposed to 0.625-20 µM doxorubicin, 40-640 nM FM19G11 and their combination (doxorubicin with and without 40-640 nM FM19G11) for 24 h. Then, the MTT solution in PBS (5 mg/mL) was replaced with the medium and incubated for 4 h. Subsequently, 100 µL dimethyl sulfoxide (DMSO) was added and absorbance was measured at 570 nm with a reference wavelength of 690 nm. The Combination Index (CI) was calculated using the Compusyn software (http://www.combosyn. com/) that measures the combination effect based on the Chou-Talalay method.

Apoptosis assay

For apoptosis assay, 5×105 cells/well were seeded into 6-well plates and incubated for 24 h. The defined concentrations of doxorubicin and FM 19G11 (concentrations that had a more synergistic effect, doxorubicin IC50 in combination with 320 nM of FM19G11 for MCF-7 in hypoxic conditions, in combination with 320 nM of FM19G11 for CSC-LCs under normoxic conditions, and in combination with 40 nM of FM19G11 for CSC-LCs under hypoxic conditions) were added. Twenty-four hours after the treatment, the cells were harvested and re-suspended in 100 µL binding buffer and stained with Annexin V (5 μL) and PI (2 µg/mL) for 15 min in the dark. The ratio of apoptotic and necrotic cells was quantified using flow cytometric analysis (BD Bioscience, San Jose, CA, USA).

Cell cycle assay

The cells were treated in the manner outlined for apoptosis assay for 24 h. The harvested cells were fixed in 70% cold ethanol for 2 h at 4 °C and incubated with 500 μL PI staining solution (Sajadian et al., 2015Sajadian S, Vatankhah M, Majdzadeh M, Kouhsari SM, Ghahremani MH, Ostad SN. Cell cycle arrest and apoptogenic properties of opium alkaloids noscapine and papaverine on breast cancer stem cells. Toxicol Mech Methods. 2015;25(5):388-95.) for 30 min at room temperature in the dark. Finally, analysis was done using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Statistical analysis

Statistical analysis was executed using the GraphPad Prism 5.01 (San Diego, CA). One-way analysis of variance followed by Turkey’s post hoc test was applied for between-group comparison.

RESULTS

Cytotoxic effect of doxorubicin and FM19G11 on MCF-7 and CSC-LCs

MTT assay was used to evaluate the cytotoxic effect of doxorubicin with or without FM19G11 on MCF-7 and CSC-LCs in normoxic and hypoxic conditions. Exposure to doxorubicin and FM19G11 showed a tendency towards MCF-7 and CSC-LCs cell proliferation suppression in a dose-dependent manner in both hypoxic and normoxic conditions (Figure 1). Our results revealed that a hypoxic condition augmented drug resistance of MCF-7 and CSC-LCs (Table I). Next, the combination effect of doxorubicin and FM19G11 on MCF-7 and CSC-LCs was evaluated. According to Figure 2, co-treatment with the above drugs in some concentrations was associated with more cytotoxic effects compared to single-drug treated cells. Furthermore, in both cells, the combination of drugs was more efficacious in hypoxic versus normoxic conditions.

FIGURE 1
Cytotoxic effect of doxorubicin and FM19G11 on MCF-7 and CSC-LCs in normoxic and hypoxic conditions. (A, C) Normoxia; (B, D) Hypoxia. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to corresponding concentration.

TABLE I
Cytotoxicity of the doxorubicin and FM19G11 against MCF-7 and CSC-LCs

FIGURE 2
Synergistic effect of doxorubicin (DOX) and FM19G11 (FM). (A) MCF-7 in normoxia; (B) MCF-7 in hypoxia; (C) CSC-LCs in normoxia; (D) CSC-LCs in hypoxia.

Although administration of doxorubicin and FM19G11 caused no synergism under normoxic conditions in MCF-7 cells, additive and synergistic effects were observed in the hypoxic condition. In CSC-LCs, 320 nM FM19G11 in combination with any concentration of doxorubicin could induce synergism cytotoxicity under normoxic conditions. This effect was also achieved for 40 and 80 nM FM19G11, and all tested concentrations of doxorubicin under hypoxic conditions. Overall, better results were achieved for combination therapy (lower CI) in CSC-LCs relative to MCF-7 cells in similar conditions.

Effect of doxorubicin and FM19G11 on MCF-7 and CSC-LCs apoptosis

Annexin V and PI staining revealed that MCF-7 cells treated with doxorubicin at the IC50 dose and 320 nM FM19G11 in hypoxic conditions underwent statistically significant late apoptotic and necrotic cell death (Figure 3). At this concentration, a marked late apoptosis (15.49% vs. 0.34%) and necrosis (13.5% vs. 0.94%) were detected in CSC-LCs compared to the control group under normoxic conditions. These percentages increased when CSC-LCs were exposed to doxorubicin IC50 and 40 nM FM19G11 in hypoxic conditions compared to doxorubicin-treated cells. Our results indicated that the combination of doxorubicin and FM19G11 fortified necrotic cell death. However, no evident increase was found in programmed cell death in CSC-LCs upon treatment with doxorubicin, implying that these cells were resistant to doxorubicin-induced apoptosis.

FIGURE 3
Combination treatment with doxorubicin (DOX) and FM19G11 (FM) resulted in apoptotic and necrotic cell death in MCF-7 and their CSC-LCs in hypoxic (H) and normoxic (N) conditions. (A) MCF-7 cells in hypoxic condition; (B) CSC-LCs in normoxic condition; (C) CSC-LCs in hypoxic condition; (D) Percentage of cells that underwent apoptosis and necrosis. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to control group.

Effect of doxorubicin and FM19G11 on cell cycle distribution of MCF-7 and CSC-LCs

Cell cycle progression was quantitatively evaluated by flow cytometric analysis (Figure 4). MCF-7 and their CSC-LCs that received combination therapy in hypoxic and normoxic conditions respectively displayed a weak accumulation in the G2/M phase. With regard to CD44+/CD24- CSC-LCs in hypoxic conditions, a combination of doxorubicin and FM19G11 could arrest cells in the G2/M phase (25% vs. 15.04% of the control group; P < 0.001) with a modest reduction in the percentage of the S phase population (30.1% vs. 42.06% of the control group).

FIGURE 4
Exposure to doxorubicin (DOX) and FM19G11 (FM) in hypoxic (H) and normoxic (N) conditions induced G2 phase cell cycle arrest. (A) MCF-7 cells in hypoxic condition; (B) CSC-LCs in normoxic condition; (C) CSC-LCs in hypoxic condition; (D) Cell cycle diagram. *P < 0.05, and **P < 0.01 as compared to control group were referred to G2/M phase.

DISCUSSION

Breast cancer is known as a systematic disease with a stem cell origin. Since the first report of BCSCs by Al-Hajj et al., (2003Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci. 2003; 100(7): 3983-8), several biomarkers have been proposed to identify them based on specific molecular markers using FACS analysis. The main markers of BCSCs are CD44+ and CD24-/low (Dittmer, 2018Dittmer J. Breast cancer stem cells: Features, key drivers and treatment options. Semin Cancer Biol. 2018;53:59-74.). In fact, CD44+/CD24- cells, which are most prevalent in basal-like/BRCA1 hereditary breast carcinoma with a triple negative state, have an essential role in proliferation, angiogenesis, and metastasis, causing poor prognosis (Giatromanolaki et al., 2011Giatromanolaki A, Sivridis E, Fiska A, Koukourakis MI. The CD44+/CD24− phenotype relates to ‘triple-negative’state and unfavorable prognosis in breast cancer patients. Med Oncol. 2011;28(3):745-52.). In addition, other markers such as ALDH1 (aldehyde dehydrogenase), ESA, CD133, CD61, and CD49f, as well as lack of expression of CD2, CD3, CD16, CD18, CD31, CD64, and CD140b are also used (Bozorgi, Khazaei, Khazaei, 2015Bozorgi A, Khazaei M, Khazaei MR. New findings on breast cancer stem cells: a review. J Breast Cancer. 2015;18(4):303-12.; Velasco-Velázquez et al., 2011Velasco-Velázquez MA, Popov VM, Lisanti MP, Pestell RG. The role of breast cancer stem cells in metastasis and therapeutic implications. Am J Pathol. 2011;179(1):2-11.). In this study, 37% of the CSC-LC population expressed CD44+/CD24- biomarkers, which is consistent with a previous study (Villadsen et al., 2007Villadsen R, Fridriksdottir AJ, Rønnov-Jessen L, Gudjonsson T, Rank F, LaBarge MA, et al. Evidence for a stem cell hierarchy in the adult human breast. J Cell Biol. 2007;177(1):87-101.).

As mentioned earlier, up-regulation of ABC transporters hinders the intracellular retention of chemotherapeutic drugs such as doxorubicin in CSCs (Moitra, 2015Moitra K. Overcoming multidrug resistance in cancer stem cells. Biomed Res Int. 2015; 2015.). On the other hand, both HIFα subtypes are the main transcriptional regulators of ABC transporters. Besides, HIF1α affects the CSC survival while HIF2α contributes to stemness maintenance (Schöning et al., 2017Schöning JP, Monteiro M, Gu W. Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia-inducible factors HIF 1α and HIF 2α. Clin Exp Pharmacol Physiol. 2017;44(2):153-61.). Our results demonstrated that doxorubicin and FM19G11 could abolish cell proliferation in MCF-7 and CSC-LCs in a dose dependent manner. Meanwhile, higher IC50 concentrations of the drugs which were observed in hypoxia and CSC-LCs, confirmed the induced therapeutic resistance. Doublier et al. (2012Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E, et al. HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer. 2012;12(1):4.) also corroborated that HIF-1α up-regulated P-gp expression accompanied by doxorubicin resistance in MCF-7 cells.

Combination therapy has been mostly used to overcome drug resistance and toxicity in multifactorial diseases like cancer or infectious diseases via affecting multiple signaling pathways (Bulusu et al., 2016Bulusu KC, Guha R, Mason DJ, Lewis RP, Muratov E, Motamedi YK, et al. Modelling of compound combination effects and applications to efficacy and toxicity: state-of-the-art, challenges and perspectives. Drug Discov Today. 2016;21(2):225-38.). Previous observations revealed the positive effect of combination therapy with chemotherapy drugs and HIF inhibitors. Samanta et al. (2014Samanta D, Gilkes DM, Chaturvedi P, Xiang L, Semenza GL. Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells. Proc Natl Acad Sci U S A. 2014;111(50):E5429-E38.) reported that a combination of paclitaxel or gemcitabine with digoxin could ameliorate tumor control and prevent chemotherapy-induced breast cancer stem cells. Moreover, MSC (Se-methylselenocysteine), as an HIF-1α inhibitor, in combination with irinotecan could sensitize human head and neck squamous cells to irinotecan in hypoxic conditions (Chintala et al., 2010Chintala S, Tόth K, Cao S, Durrani FA, Vaughan MM, Jensen RL, et al. Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1α. Cancer Chemother Pharmacol. 2010;66(5):899-911.). It should be noted that a combination of HIF inhibitors with other drugs could be used clinically, such as the use of temsirolimus (an mTOR/HIF inhibitor) combined with bevacizumab and liposomal doxorubicin in a phase I clinical trial (Moroney et al., 2012Moroney J, Fu S, Moulder SL, Falchook GS, Helgason T, Levenback CF, et al. Phase I study of the anti-angiogenic antibody bevacizumab and the mTOR/hypoxia-inducible factor inhibitor temsirolimus combined with liposomal doxorubicin. Clin Cancer Res. 2012:18(20):5796-805.).

In 1984, Paul Talalay presented the scientific term “CI: combination index” synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1) (Chou, Talalay, 1984Chou T-C, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27-55.). Nowadays, several computational programs are available for CI calculation such as the Compusyn (Chou, 2006Chou T-C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58(3):621-81.). Here, the MTT results of concurrent treatment with different concentrations of doxorubicin and FM19G11 justified the calculated CIs using the Compusyn. It was also demonstrated that treatment with FM19G11 increased the efficacy of doxorubicin against CSC-LCs under hypoxic conditions. The resulting data were in accordance with a recent report that a combination of FM19G11 and temozolamide (TMZ) in glioblastomas could reverse resistance in MGMT (O6- methylguanine DNA-methyltransferase) positive cells by fortifying the pro-apoptotic effect of TMZ (You et al., 2018You Cg, Sheng Hs, Xie Cr, Zhang N, Zheng Xs. FM19G11 inhibits O6-methylguanine DNA-methyltransferase expression under both hypoxic and normoxic conditions. Cancer Med. 2018;7(7):3292-300.). In order to explore the growth inhibitory mechanism of doxorubicin and FM19G11, the treated cells were subjected to flow cytometric analysis. It was observed that co-administration of FM19G11 and doxorubicin resulted in 21% total apoptosis and 15% necrotic cells with G2/M arrest in CSC-LCs under hypoxic conditions. Previous investigations found that doxorubicin resulted in autophagy and necrosis by hyperactivation of PARP-1 (poly (ADP-ribose) polymerase-1) in response to DNA damage (Minotti et al., 2004Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev . 2004;56(2):185-229.; Tacar, Sriamornsak, Dass, 2013Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol. 2013;65(2):157-70.). Nevertheless, Huang et al. (2012Huang J, Liu K, Yu Y, Xie M, Kang R, Vernon PJ, et al. Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma. Autophagy. 2012;8(2):275-7.) claimed that autophagy is one of the reasons for doxorubicin resistance so that suppression of HMGB1 by shRNA in osteosarcoma decreases autophagy and increases sensitivity to doxorubicin.

CONCLUSION

A combination of doxorubicin and FM19G11 in MCF-7 and CD44+/CD24- cells showed synergism in hypoxic conditions and increased the cytotoxic effects of doxorubicin. This combination resulted in a G2/M arrest in the hypoxic condition in both cells and fortified apoptosis in MCF-7 and CD44+/CD24- CSC-LCs in both conditions. The results disclosed here demonstrated that HIF inhibitors in combination with cytotoxic agents could decrease the drug resistance and increase the therapeutic response.

ACKNOWLEDGMENT

The authors would like to express their gratitude to the Deputy of Research, Tehran University of Medical Science, for financial support under grant number 94-04-33-31024.

REFERENCES

  • Alastrue-Agudo A, Rodriguez-Jimenez FJ, Mocholi EL, De Giorgio F, Erceg S, Moreno-Manzano V. FM19G11 and ependymal progenitor/stem cell combinatory treatment enhances neuronal preservation and oligodendrogenesis after severe spinal cord injury. Int J Mol Sci. 2018;19(1):200.
  • Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci. 2003; 100(7): 3983-8
  • Bhattarai D, Xu X, Lee K. Hypoxia-inducible factor-1 (HIF-1) inhibitors from the last decade (2007 to 2016): A “structure-activity relationship” perspective. Med Res Rev. 2017;38(4):1404-42.
  • Bozorgi A, Khazaei M, Khazaei MR. New findings on breast cancer stem cells: a review. J Breast Cancer. 2015;18(4):303-12.
  • Bulusu KC, Guha R, Mason DJ, Lewis RP, Muratov E, Motamedi YK, et al. Modelling of compound combination effects and applications to efficacy and toxicity: state-of-the-art, challenges and perspectives. Drug Discov Today. 2016;21(2):225-38.
  • Burroughs SK, Kaluz S, Wang D, Wang K, Van Meir EG, Wang B. Hypoxia inducible factor pathway inhibitors as anticancer therapeutics. Future Med Chem. 2013;5(5):553-72.
  • Chintala S, Tόth K, Cao S, Durrani FA, Vaughan MM, Jensen RL, et al. Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1α. Cancer Chemother Pharmacol. 2010;66(5):899-911.
  • Chou T-C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58(3):621-81.
  • Chou T-C, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27-55.
  • Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer. 2005;5(4):275-84.
  • Dittmer J. Breast cancer stem cells: Features, key drivers and treatment options. Semin Cancer Biol. 2018;53:59-74.
  • Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E, et al. HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer. 2012;12(1):4.
  • Economopoulou P, Kaklamani VG, Siziopikou K. The role of cancer stem cells in breast cancer initiation and progression: potential cancer stem cell-directed therapies. Oncologist. 2012;17(11):1394-401.
  • El Assar M, Sanchez-Puelles JM, Royo I, Lopez-Hernandez E, Sanchez-Ferrer A, Acena JL, et al. FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway, independently of mTOR/HIF-1α activation. Br J Pharmacol. 2015;172(5):1277-91.
  • Giatromanolaki A, Sivridis E, Fiska A, Koukourakis MI. The CD44+/CD24− phenotype relates to ‘triple-negative’state and unfavorable prognosis in breast cancer patients. Med Oncol. 2011;28(3):745-52.
  • Huang J, Liu K, Yu Y, Xie M, Kang R, Vernon PJ, et al. Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma. Autophagy. 2012;8(2):275-7.
  • Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev . 2004;56(2):185-229.
  • Moitra K. Overcoming multidrug resistance in cancer stem cells. Biomed Res Int. 2015; 2015.
  • Moreno-Manzano V, Rodríguez-Jiménez FJ, Aceña-Bonilla JL, Fustero-Lardíes S, Erceg S, Dopazo J, et al. FM19G11, a new hypoxia-inducible factor (HIF) modulator, affects stem cell differentiation status. J Biol Chem. 2010;285(2):1333-42.
  • Moroney J, Fu S, Moulder SL, Falchook GS, Helgason T, Levenback CF, et al. Phase I study of the anti-angiogenic antibody bevacizumab and the mTOR/hypoxia-inducible factor inhibitor temsirolimus combined with liposomal doxorubicin. Clin Cancer Res. 2012:18(20):5796-805.
  • Nassar D, Blanpain C. Cancer stem cells: basic concepts and therapeutic implications. Annu Rev Pathol-Mech. 2016;11(1):47-76.
  • Phillips TM, McBride WH, Pajonk F. The response of CD24−/low/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst. 2006;98(24):1777-85.
  • Sajadian S, Vatankhah M, Majdzadeh M, Kouhsari SM, Ghahremani MH, Ostad SN. Cell cycle arrest and apoptogenic properties of opium alkaloids noscapine and papaverine on breast cancer stem cells. Toxicol Mech Methods. 2015;25(5):388-95.
  • Samanta D, Gilkes DM, Chaturvedi P, Xiang L, Semenza GL. Hypoxia-inducible factors are required for chemotherapy resistance of breast cancer stem cells. Proc Natl Acad Sci U S A. 2014;111(50):E5429-E38.
  • Schöning JP, Monteiro M, Gu W. Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia-inducible factors HIF 1α and HIF 2α. Clin Exp Pharmacol Physiol. 2017;44(2):153-61.
  • Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE. Hypoxia-inducible nuclear factors bind to an enhancer element located 3’ to the human erythropoietin gene. Proc Natl Acad Sci U S A. 1991;88(13):5680-4.
  • Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol. 2013;65(2):157-70.
  • Theriault RL, Carlson RW, Allred C, Anderson BO, Burstein HJ, Edge SB, et al. Breast cancer, version 3.2013. J Natl Compr Canc Netw. 2013;11(7):753-61.
  • Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87-108.
  • Velasco-Velázquez MA, Popov VM, Lisanti MP, Pestell RG. The role of breast cancer stem cells in metastasis and therapeutic implications. Am J Pathol. 2011;179(1):2-11.
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Publication Dates

  • Publication in this collection
    22 Apr 2022
  • Date of issue
    2022

History

  • Received
    16 Sept 2018
  • Accepted
    12 July 2019
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br