| Objective:Breast cancer(BC)is one of the most common malignant tumors among women in the world,and its incidence rate and mortality have shown a significant upward trend.Paclitaxel(PTX)is the basic drug for early neoadjuvant chemotherapy,postoperative adjuvant chemotherapy,and metastatic breast cancer treatment of breast cancer.However,it can cause neurotoxicity and other adverse reactions while treating breast cancer,which seriously limits its clinical application.DKK1 is a member of the Dickkopf family,which can not only inhibit the infiltration of cytotoxic T lymphocytes and NK cells to mediate tumor immune escape but also induce the loss of dendritic spines in primary cortical neurons in rats,leading to nerve damage.Whether paclitaxel can induce DKK1 expression has not been reported.1.To explore whether paclitaxel mediates the expression of DKK1 in breast cancer cells,clinical specimens and mouse transplanted tumor models were used to investigate the expression of DKK1 in tumor tissue after paclitaxel chemotherapy:Immunohistochemical staining was used to detect the expression of DKK1 in breast cancer clinical specimens and mouse tumor slices before and after paclitaxel chemotherapy;Real-time PCR and Western blot were used to detect the expression of DKK1 mRNA and protein in Four types of breast cancer cell lines(MCF-7,MDA-MB-231,BT474,SK-BR-3)were cultured with 200 n M paclitaxel;detection of DKK1 content in breast cancer cell culture medium after paclitaxel incubation by ELISA.2.To explore the effect of DKK1 on the growth of breast cancer and the infiltration of immune cells in tumor tissues of mice by using a mouse model of transplanted tumor:Construct 4T1 cell lines with DKK1overexpression and knockdown,use them to establish a mouse solid tumor model,and observe the effect of DKK1 overexpression and knockdown on the growth of subcutaneous transplanted tumors in mice.the infiltration of CD3~+,CD8~+,and GZMB~+immune cells in mouse tumor tissue was detected by flow cytometry.3.To explore whether paclitaxel mediates the expression of EGFR in breast cancer cells,using clinical specimens and mouse transplanted tumor models to investigate the expression of EGFR in tumor tissue after paclitaxel chemotherapy:Immunohistochemical staining was used to detect the expression of EGFR in clinical specimens of breast cancer and tumor sections of mice before and after paclitaxel chemotherapy;western blot assay was used to detect the expression of p-EGFR and EGFR protein in two different breast cancer cell lines(MCF-7,MDA-MB-231)were incubated with 200 n M paclitaxel.4.To explore whether EGFR mediates DKK1 transcription in breast cancer cells:Real-time PCR and Western blot were used to detect the expression of DKK1 mRNA and protein in two breast cancer cell lines(MCF-7 and MDA-MB-231)of EGFR overexpressed;Real-time PCR and Western blot were used to detect the expression of DKK1 mRNA and protein in breast cancer cells of were incubated with paclitaxel while AG1478.5.To investigate whether paclitaxel mediates DKK1 transcription in mouse DRG:The mechanical pain and thermal pain thresholds of mice treated with paclitaxel were measured using the Von Frey hair method and a hot plate instrument;Effects of paclitaxel on the morphology and structure of neurons in the sciatic nerve of mice detected by transmission electron microscopy;immunofluorescence staining and Western blot assay were used to detect the expression of PGP9.5 in mouse hind paw skin tissue to explore the effect of paclitaxel on nerve fiber density;Real-time PCR and Western blot experiments were used to detect the expression of DKK1 mRNA and protein in DRG of mice treated with paclitaxel.6.To investigate whether DKK1 is involved in sciatic neuropathy and peripheral nerve injury in the nervous system of mice:In the establishment of a mouse solid tumor model with DKK1 overexpression and knockdown 4T1 cell lines and the establishment of an animal model with intrathecal injection of LV-DKK1-OE/LV-sh-DKK1,the mechanical and thermal pain thresholds in mice were measured using the Von Frey hair method and a hot plate instrument;detection of the effect of DKK1 on the morphology and structure of neurons in the sciatic nerve of mice by transmission electron microscopy;to explore the effect of DKK1 on nerve fiber density,immunofluorescence staining and Western blot assay were used to detect the expression of PGP9.5 protein in the skin tissue of the hind foot of mice.7.Establish a solid tumor animal model to investigate whether EGFR inhibitors enhance the antitumor effect of paclitaxel and reduce its induced neurotoxicity by inhibiting the expression of DKK1 in mouse tumor tissue and DRG:Using 4T1 cell line to establish a mouse subcutaneous transplanted tumor model,paclitaxel and AG1478 were administered simultaneously to observe the effect of AG1478 combined with paclitaxel on the growth of subcutaneously transplanted tumor in mice;detection of mechanical pain and thermal pain thresholds in mice using the Von Frey hair method and a hot plate instrument;Real-time PCR and Western blot experiments were used to detect the expression of DKK1 mRNA and protein in mice tissue and DRG;detection of its effect on the morphology and structure of neurons in the sciatic nerve of mice by transmission electron microscopy;western blot assay was used to detect the expression of PGP9.5 protein in the hind foot of mice.8.To explore whether DKK1 neutralizing antibody combined with paclitaxel has the effect of"enhancing efficacy and reducing toxicity"in the treatment of breast cancer:Using 4T1 cell line to establish a subcutaneous transplanted tumor model in mice,paclitaxel and DKK1 neutralizing antibody were administered simultaneously to detect their effects on the growth of subcutaneous transplanted tumors in mice;detection of mechanical pain and thermal pain thresholds in mice using the Von Frey hair method and a hot plate instrument;the effects of DKK1 neutralizing antibody on the morphology and structure of neurons in the sciatic nerve of mice were examined by transmission electron microscopy;Western blot assay was used to detect the expression of PGP9.5 protein in the hind foot of mice.Results:1.In clinical specimens of breast cancer and tumor tissue sections of mice,the expression of DKK1 in tumor tissue of patients receiving paclitaxel chemotherapy significantly increased;after paclitaxel was given to incubate four different breast cancer cell lines(MCF-7,MDA-MB-231,BT474,SK-BR-3),the expression levels of DKK1 mRNA and protein were significantly up-regulated;ELISA experiments showed that the secretion of DKK1 into cell culture medium significantly increased after paclitaxel stimulation.2.Overexpression of DKK1 can significantly promote tumor growth in a mouse solid tumor model;overexpression of DKK1 significantly reduces the infiltration of CD3~+,CD8~+,and GZMB~+immune cells in tumor tissue;knocking down DKK1 inhibits tumor growth and enhances the infiltration of CD3~+,CD8~+,and GZMB~+immune cells in tumor tissue.3.In clinical specimens of breast cancer and tumor tissue sections of mice,the expression of EGFR in tumor tissue of patients receiving paclitaxel chemotherapy significantly increased;the expression levels of p-EGFR and EGFR proteins were significantly upregulated after incubating MCF-7 and MDA-MB-231 cells with paclitaxel.4.Overexpression of EGFR can significantly promote the expression of DKK1 mRNA and protein in MCF-7 and MDA-MB-231 breast cancer cells;the expression of DKK1 mRNA and protein induced by paclitaxel was significantly reversed after incubation with AG1478.5.The mechanical pain and thermal pain thresholds in mice were significantly reduced after receiving paclitaxel treatment;the sciatic nerve in mice showed significant axonal swelling,myelin sheath loosening,and rupture after treatment with paclitaxel;the expression of PGP9.5 in the epidermis of the mouse hind foot decreased,and the density of nerve fibers significantly decreased;the expression of DKK1 in DRG of mice increased significantly after paclitaxel treatment.6.Overexpression of DKK1 induces a decrease in mechanical and thermal pain thresholds in mice;Overexpression of DKK1 can induce axonal swelling,myelin sheath rupture,and significantly inhibit the expression of PGP9.5 protein in mouse hind foot skin tissue;knocking down DKK1 can reverse these phenomena.7.AG1478 significantly enhances the antitumor effect of paclitaxel and reverses the decrease in mechanical and thermal pain thresholds induced by paclitaxel in mice;AG1478 inhibits the expression of DKK1 mRNA and protein in mouse tumor tissue and DRG;AG1478 reversed the neuropathy induced by paclitaxel and significantly reversed the inhibitory effect of paclitaxel on PGP9.5 protein in mouse hind foot skin tissue,enhancing the density of nerve fibers in mice.8.DKK1 neutralizing antibody significantly increased the anti-tumor effect of paclitaxel,reversing the decrease in mechanical and thermal pain thresholds induced by paclitaxel in mice;DKK1 neutralizing antibody reversed paclitaxel induced neuropathy and significantly reversed the inhibitory effect of paclitaxel on PGP9.5 protein in mouse hind foot skin tissue.Conclusions:Paclitaxel induces the expression of EGFR in breast cancer cells,and then mediates the transcription of DKK1,causing immune escape of breast cancer cells and peripheral nerve injury;DKK1 neutralizing antibody enhances the anti-breast cancer effect of paclitaxel by antagonizing the molecular function of DKK1and alleviates the nerve injury induced by paclitaxel. |
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