| 1.BackgroundIn recent years,the incidence rate of breast cancer in the world is increasing,which is a great threat to women’s physical and mental health and even life.Pain is one of the most common clinical symptoms in patients with advanced breast cancer.The incidence rate of pain in patients with advanced breast cancer is as high as 70%～90%.Cancer pain reduces the quality of life of tumor patients in different degrees from physiological,psychological,spiritual and social aspects.According to the three-step treatment plan for cancer pain recommended by the World Health Organization(WHO),opioid analgesics represented by morphine are still the most effective analgesic drugs for cancer pain.However,current research shows that morphine,fentanyl and other commonly used clinical opioids can promote the proliferation of tumor stem cells(CSCs)in vitro,improve the expression of CSCs-related molecules OCT4,SOX2 and NANOG,and reduce the sensitivity of chemotherapy drugs,thus increasing the tumor recurrence rate and shortening the survival time of tumor patients after surgery.Therefore,the choice of analgesic drugs is directly related to the quality of life and survival rate of breast cancer patients,and it is of great significance and necessity to deeply explore therapeutic schemes that can effectively control cancer pain and inhibit tumor growth and metastasis.Nalbuphine is an opioid receptor agonist-antagonist analgesic,which can be used to relieve various moderate and severe pains.In terms of mass units,the analgesic effect of nalbuphine is basically equivalent to that of morphine,with fewer adverse reactions.Although nalbuphine has been proved to be an effective and reliable analgesic choice,its relationship with tumor development has not received much attention.With the continuous rise of the theory of tumor stem cells,our cognition of tumor stem cells is gradually profound.Tumor stem cells are a very small group of cells with self-renewal and multidirectional differentiation potential in tumors,which play an important role in the survival,proliferation,recurrence and metastasis of tumors.At the same time,a large number of genetic and cellular biological evidences show that AKT-NFκB pathway can be activated by many growth factors and biological stimuli,and is closely related to tumor stemness.Therefore,whether nalbuphine can reduce the stemness of breast cancer and slow down the development of breast cancer by inhibiting AKT-NFκB pathway,thus providing a new intervention scheme for clinical treatment of breast cancer pain,has become the focus of our research.2.Methods(1)(1)MTT assay was used to detect the survival and growth of breast cancer cells(MDA-MB-231,MCF-7,SK-BR-3)after nalbuphine treated.(2)The effect of nalbuphine on the proliferation of breast cancer cells(MDA-MB-231,MCF-7,SK-BR-3)was detected by colony formation assays.(3)MTT assay was used to detect the effect of nalbuphine on the proliferation of other tumor cells(MDA-MB-231,MCF-7,SK-BR-3,A549,NCI-H460,NCI-H1299,Hep G2,AGS,HCT116,He La,TT,CNE1)and normal breast cells(MCF-10 A,MCF-10F).(2)(1)We collected samples of breast cancer cells(MDA-MB-231,MCF-7,SK-BR-3)treated by nalbuphine,and used Western blot and RT-PCR to detect the effect of nalbuphine on the expression of self-renewal markers(SOX2,OCT4,NANOG,MYC).(2)The ALDH1-positive sorting assay was used to analyze the effect of nalbuphine on breast cancer stem-like properties.(3)The effect of nalbuphine on the sphere formation ability of breast cancer cells(MDA-MB-231)was detected by limiting dilution assay and sphere formation assay.(4)Western blot was used to detect the effect of nalbuphine on the expression of self-renewal markers in normal stem cells(MSC).(3)(1)The mouse xenograft assay was used to verify the effect of nalbuphine on tumor development(5 mice in each group),and the tumor volumes of each group were compared after 30 days of culture.(2)The liver and kidney toxicity of nalbuphine to mice was verified by ELISA.(3)We collected tumor tissues from mice and primary xenografts were subjected to sphere formation assay,Western blot and IHC to verify the effect of nalbuphine on breast cancer stem-like properties.(4)The effect of nalbuphine on the cancer stem-like traits of breast cancer was further verified by in vivo serial dilution assay.(5)Samples of breast cancer cells(MDA-MB-231,MCF-7)treated by nalbuphine,morphine and fentanyl were collected,and the effects of nalbuphine,morphine and fentanyl on the expression of self-renewal markers were compared by Western blot.(6)The mouse xenograft assay was used to compare the effect of nalbuphine,morphine and fentanyl on tumor development(3 mice in each group),and after 30 days of culture,the tumor volumes of each group were compared.(4)(1)RT-q PCR and Western blot were used to detect the effect of nalbuphine on the expression of EMT-related markers(E-cadherin,N-cadherin,Vimentin,Snail)in breast cancer cells(MDA-MB-231,MCF-7,SK-BR-3).(2)Wound healing and transwell assays were used to detect the effect of nalbuphine on the migration and invasion of breast cancer cells(MDA-MB-231,MCF-7,SK-BR-3).(3)Tumors extracted from nalbuphine-treated mice were also used Western blot and IHC to verify the effect of nalbuphine on breast cancer metastasis.(5)(1)Western blot was used to detect the effect of nalbuphine on AKT-NFκB pathway.(2)Akt phosphorylation activators SC79 and IGF-1 were used,and Western blot,sphere formation assay,the mouse xenograft assay and wound healing assay were used to verify the effect of nalbuphine on AKT-NFκB pathway.3.Results(1)(1)MTT assay showed that nalbuphine could effectively inhibit the proliferation of breast cancer cells in a dose-and time-dependent manner.(2)The results of colony formation assays showed that nalbuphine can effectively inhibit the proliferation of breast cancer cells,which is dose-related.(3)MTT assay showed that nalbuphine had inhibitory effect on proliferation of many other different types of tumor cells,but had no obvious inhibitory effect on proliferation of normal breast cells.(2)(1)Western blot and RT-PCR results showed that nalbuphine inhibited the expression of self-renewal markers in breast cancer cells.(2)The ALDH1-positive sorting assay showed that nalbuphine inhibited breast cancer stem-like properties.(3)The results of limiting dilution assay and sphere formation assay showed that nalbuphine inhibited the sphere formation ability of breast cancer cells.(4)Western blot results showed that nalbuphine had no obvious effect on self-renewal markers of normal stem cells.(3)(1)Results of the mouse xenograft assay showed that nalbuphine could inhibit subcutaneous tumor formation in mice.(2)ELISA results showed that nalbuphine had no obvious liver and kidney toxicity to mice.(3)Sphere formation assay,Western blot and IHC results showed that nalbuphine inhibited the sphere formation ability of primary cells of tumor tissues and the expression of self-renewal markers in tumor tissues.(4)In vivo serial dilution assay showed that nalbuphine inhibited breast cancer stem-like properties.(5)Western blot results showed that nalbuphine inhibited the expression of self-renewal markers in breast cancer cells,while morphine and fentanyl promoted the expression of self-renewal markers in breast cancer cells.(6)The mouse xenograft assay showed that nalbuphine could inhibit subcutaneous tumor formation in mice,while morphine and fentanyl could promote subcutaneous tumor formation in mice.(4)(1)RT-q PCR and Western blot results showed that nalbuphine inhibited the expression of EMT-related markers in breast cancer cells.(2)Wound healing and transwell assays results showed that nalbuphine had inhibitory effect on the migration and invasion of breast cancer cells.(3)Western blot and IHC results of tumor tissues showed that nalbuphine inhibited breast cancer metastasis.(5)(1)Western blot results showed that nalbuphine inhibited AKT-NFκB pathway activity.(2)The Akt phosphorylation activator SC79 and IGF-1 were used,and results of Western blot,sphere formation assay,the mouse xenograft assay and wound healing assay showed that nalbuphine inhibited breast cancer stem-like properties through AKT-NFκB pathway.4.Conclusions(1)Nalbuphine inhibits the proliferation of breast cancer cells in a dose-and time-dependent manner.(2)Nalbuphine inhibits breast cancer stem-like properties in vitro.(3)Nalbuphine inhibits breast cancer stem-like properties in vivo.(4)Nalbuphine inhibits EMT and metastasis of breast cancer.(5)Nalbuphine inhibits breast cancer stem-like properties,EMT and metastasis through AKT-NFκB pathway. |
PDF Full Text Request