DOXIL when combined with Withaferin A (WFA) targets ALDH1 positive cancer stem cells in ovarian cancer.

Ovarian cancer is a highly aggressive and deadly disease. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly cisplatin or carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately, after few months of initial treatment, tumor relapse occurs due to platinum-resistance. DOXIL (liposomal preparation of doxorubicin) is a choice of drug for recurrent ovarian cancer. However,itsresponserateisverylowandisaccompaniedbymyocardialtoxicity.Resistancetochemotherapyandrecurrenceof cancer is primarily attributed to the presence of cancer stem cells (CSCs), a small population of cells present in cancer. Effect of DOXIL and withaferin A (WFA), both alone and in combination, was investigated on cell proliferation of ovarian cancer cell line A2780 and tumor growth in SCID mice bearing i.p. ovarian tumors. ALDH1 cells were isolated from A2780 using cell sorter, and effectofDOXILandWFAbothaloneandincombinationontumorigenicfunctionofALDH1wasstudiedusingspheroidsformation assaysinvitro.WesternblotswereperformedtoexaminetheexpressionofALDH1andNotch1genes.Inourstudies,weshowed, for the first time, that DOXIL when combined with withaferin A (WFA) elicits synergistic effect on inhibition of cell proliferation of ovarian cancer cells and inhibits the expression of ALDH1 protein, a marker for ALDH1 positive cancer stem cells (CSCs), and Notch1,asignalingpathwaygenerequiredforself-renewalofCSCs.InhibitionofexpressionofbothALDH1andNotch1genesby WFA was found to be dose dependent, whereas DOXIL (200 nM) was found to be ineffective. SCID mice, bearing i.p. ovarian tumors, were treated with a small dose of DOXIL (2 mg/kg) in combination with a sub-optimal dose of WFA (2 mg/kg), which resultedinahighlysignificant(60%to70%)reductionintumorgrowthandcompleteinhibitionofmetastasiscomparedtocontrol. In contrast, WFA treatment showed a significant reduction in tumor growth but no change in metastasis compared to control. DOXIL showed non-significant reduction in tumor growth and no change in metastasis compared to control. Isolated ALDH1 positive CSCs treated with the combination of DOXIL and WFA resulted in a significant reduction in spheroids formation (tumorigenicfunctionofCSCs)andexpressionofALDH1protein.WFAwhenusedaloneataconcentrationof1.5mMwasfoundto behighlyeffectiveinsuppressionofALDH1expression,whereasDOXILataconcentrationof200nMwasfoundtobeineffective. DOXIL in combination with WFA elicits synergistic effects, targets cancer stem cells, and has potential to minimize induction of drug resistanceandreoccurrenceofcancer.Basedonourstudies,weconcludethatthecombinationofDOXILwithWFAhasthe potential to be an effective therapy for ovarian cancer and may ameliorate DOXIL related side effects as well as recurrence of ovarian cancer leading to increase in patients' survival rate.