Morphine Promotes Cancer Stem Cell Properties, Contributing To Chemoresistance In Breast Cancer

Objective: Breast cancer stem cells(BCSCs) contain a subpopulation of CD44+/CD24-/low and self-renewing cells in breast cancer. BCSCs are more resistant to traditional chemotherapy than non-BCSCs, but the molecular mechanisms by which CSCs contribute to drug resistance remain to be determined, Thus targeting CSCs is the current challenge for the development of anti-cancer treatment.Morphine is an opioid analgesic drug, which imitates endogenous enkephalin and excites the receptors of central nervous system to relief pain. Morphine has been extensively used for anesthetic pre-medication and management of cancer pain with cancer metastasis. whereas, both morphine and anticancer drugs have been simultaneously given to patients Currently, especially those patients with cancer metastasis. However, emerging evidence showed that morphine had extra analgesic effects that appeared to alter tumor progression by activating non-classical opioid receptor signaling. Morphine induces phosphorylation of epidermal growth factor receptor(EGFR) via opioid receptors, promotes cell proliferation and increases cell invasion. Morphine also activates MAPK/ERK by phosphorylation via PTX-sensitive GPCRs and NO, which leads to the promotion of tumor growth in breast cancer. In addition, morphine promotes breast cancer cell migration and invasion by increasing the expression of NET1. So the mechanism of morphine promoting cancer progression is widely under attention.Here, we report that morphine enhances the mammosphere forming capacity and increases the expression of stemness-related transcription factors Oct4, Sox2 and Nanog.Treatment with morphine leads to enrichment of a side population fraction in MCF-7cells and the CD44+/CD24-/low population in BT549 cells. Consistently, morphine activates Wnt/β-catenin signaling to induce epithelial to mesenchymal transition and promotes metastasis. Moreover, morphine decreases the sensitivity of traditional anti-cancer drugs in breast cancer cells. Nalmefene, an antagonist of morphine, reverses morphine-induced cancer stem cell properties and chemoresistance in breast cancer. In addition, nalmefene abolishes morphine enhancing tumorigenesis in a NOD/SCID mouse model. In conclusion, our findings demonstrate that morphine contributes to chemoresistance via expanding the population of cancer stem cells and promotes tumor growth, thereby revealing a novel role of morphine and providing some new guides in clinical use of morphine.Methods: 1. Mammosphere formation assay was used to detective the stemness breast cancer cells treated with morphine. Cancer stem cell surface marker CD44+/CD24-/low and side population assay via pumping Hoechst 33342 to determine the effect of morphine to cancer stem cells.2. Quantitative Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) and western blot were used to evaluate the relative expression quantity of stem-like marker Oct4, Sox2 and Nanog.3. The mRNA relative level of CDH1,CDH2,CTNNB1 in EMT signal pathway was measured by RT-PCR of BT549 cells treated with morphine(0, 1, 10 μM) for 4days. Western blot determines protein level of E-cadherin,N-cadherin,β-catenin and the location via immunofluorescence staining. Nuclear/Cytosol Fractionation assay was used to determine the level of β-catenin in nuclear. In the migration and invasion analysis, the acceerative effects of morphine on invasive and migratory capabilities were evaluated by Transwell assay.4. BT549 cells were pretreated with morphine for 4 days, followed by incubated with doxorubicin(0.5 μM) or paclitaxel(10 nM) for another 2 days. The facilitatedeffect of different concentration of morphine to BT549 cells and whether morphine was able to reduce the sensitivity of BT549 cells to chemotherapy drug doxorubicin or paclitaxel were evaluated by MTT assay. The effect of morphine on doxorubicin or paclitaxel induced apoptosis in breast cancer cells was assessed through cleaved PARP and caspase-3 by western blot.5. MTT assay was used to detect whether nalmefene was able to reverse the facilitated effect of morphine to breast cancer cell BT549 and sensitivity to doxorubicin or paclitaxel. It also was determined by western blot assay and sphere formation assay.6. The effect of morphine and nalmefene to tumorigenesis in vivo was evaluated by mouse xenograft assay.Results: 1. Morphine significantly increased the sphere size and number in both MCF-7 cells and BT549 cells. For side population assay, side population fraction increased from 0.63 ± 0.30 % to 3.4 ± 0.15 % compared with the untreated group in MCF-7 cells.2. The mRNA levels of Oct4, Sox2 and Nanog in MCF-7 and BT549 cells treated with morphine by RT-PCR. Morphine significantly increased the mRNA levels of Oct4,Sox2 and Nanog in both MCF-7 and BT549 cells. Western blot assay showed that morphine dose dependent increased the protein levels of Oct4, Sox2 and Nanog in MCF-7 and BT549 cells.3. Morphine decreased the mRNA level of CDH1 but increased the mRNA levels of CDH2 and CTNNB1 in both MCF-7 and BT549 cells. Consistently, morphine decreased the expression of E-cadherin but increased the expression of N-cadherin and?-catenin. Moreover, the immunofluorence staining results also showed that morphine decreased the expression of E-cadherin while increased the expression of N-cadherin and ?-catenin in both MCF-7 and BT549 cells. Nuclear/Cytosol Fractionation assay showed level of β-catenin in nuclear is up-regulated. Transwell assay showed that morphine could significantly enhance cell migration and invasion abilities in BT549 cells.4. BT549 cells were pretreated with morphine for 4 days, followed by incubatedwith doxorubicin(0.5 μM) or paclitaxel(10 nM) for another 2 days. Morphine alone did not apparently alter the cell viability, but significantly abolished the loss of cell viability induced by doxorubicin or paclitaxel. Western blot results showed that doxorubicin or paclitaxel induced the cleavage of PARP and caspase-3, but were apparently recovered by morphine.5. MTT, Western blot and apoptosis assay showed nalmefene could reverse the effect of morphine in both sphere forming ability and chemoresistance to doxorubicin or paclitaxel.6. NOD/SCID mouse modle showed morphine promotes tumorigenesis which can be reversed by nalmefene.Conclusions: The studies showed morphine could promote tumorgensis, EMT,metastasis and invasion, contribute to chemoresistance in breast cancer, and these effects were revered by nalmefene.