Morphine Promotes Breast Cancer Cells Proliferation, Contributing To Chemoresistance In Breast Cancer

Objective: 1. To explore the effect of morphine on cell proliferation, sphereforming ability in MCF-7 and MCF-10 A cells. To determine whether morphine cause chemotherapy resistance in breast cancer cells. To investigate whether nalmefene could inverse the effect of morphine.Methods: 2. Mammosphere formation assay was used to detective the stemness of breast cancer cells treated with morphine. Colony formation assay was used to test the effect of morphine in proliferation. 3. Quantitative Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) and Western blot assay were used to evaluate the relative expression quantity of Oct4 and C-myc. 4. The effect of different concentration of morphine in MCF-7 cells and whether morphine was able to reduce the sensitivity of MCF-7 cells to doxorubicin or paclitaxel were evaluated by MTT assay. The effect of morphine on doxorubicin or paclitaxel induced apoptosis in MCF-7 cells was assessed through cleaved Caspase-3 and cleaved PARP by Flow cytometry and Western blot assay. 5. Sphereforming ability was used to determine the effect of nalmefene in reversing morphine’s effect of promoting cancer cells proliferation.Results: 1. Morphine promoted MCF-7 and MCF-10 A cells sphereforming ability. In comparison to those of untreated controls, Morphine significantly increased the sphere size and number in both MCF-7 cells and MCF-10 A cells(P < 0.001). 2. Morphine also increased colony formation rate(P < 0.001) in both MCF-7 and MCF-10 A cells. 1μM morphine colony formation rate increased to 20.14 % ±1.32 %, 18.06 % ± 0.76 %(P < 0.01). In consistant,10 μM morphine treated cells, colony foramation rate increased to 32.48 % ± 0.85 %, 27.54 % ±0.76 %(P < 0.001). 3. We determined the m RNA levels of Oct4, C-myc in MCF-7 treated with morphine by q RT-PCR. Morphine significantly increased the m RNA levels of Oct4, C-myc in MCF-7 cells. In comparison to those of untreated controls, the m RNA levels of Oct4, C-myc were increased respectively by 2.4 ± 0.15(P < 0.01), 13.6 ±0.21(P < 0.001) and 4.3±0.13(P <0.01), 15.7 ± 0.16(P < 0.001). In consistent, Western blot assay showed that morphine dose dependent increased the protein levels of Oct4 and C-myc in MCF-7. 4. Morphine significantly abolished the loss of cell viability induced by doxorubicin or paclitaxel. Western blot results showed that 0.5 M doxorubicin or 10 n M paclitaxel induced the cleavage of PARP and caspase-3, but were apparently recovered(1 μM, 10 μM) by morphine. Compared to the controls, the apotosis rate of MCF-7 cells decreased to 19.73 % ± 0.86 %(P < 0.001), 22.23 % ± 0.50 %(P < 0.001) and 7.43 % ± 0.76 %(P < 0.001), 10.76 % ± 0.56 %(P < 0.01). 5. Sphereforming assay showed that nalmefene(10 μM) could reverse the effect of morphine(10 μM) in sphere forming ability(P < 0.001).Conclusion: Morphine promotes MCF-7 and MCF-10 A cells sphereforming ability and colony formation rate. Morphine increases the expressiong of Oct4 and C-myc in both protein and m RNA level. Morphine induces chemotherapy resistance in MCF-7 cells. Nalmefene partyly reverses the effect of morphine in phere forming ability.