NK007 Helps In Mitigating Paclitaxel Resistance Through P38MAPK Activation And HK2 Degradation In Ovarian Cancer

Background: The incidence of ovarian cancer ranks second among malignant genital tumors,which poses a serious threat in women’s health.In clinical research,there are three types of ovarian cancer: germ cell,stromal,and epithelial tumors.Epithelial ovarian cancer has high incidence and mortality rate among patients and,at the onset of symptoms,70% of the patients are diagnosed with advanced cancer with metastasis and poor prognosis.Currently,the standard treatment for ovarian cancer primarily includes surgical cytoreduction and adjuvant chemotherapy with paclitaxel(PTX)and carboplatin or cisplatin.However,chemoresistance emerges frequently and contributes to high recurrence rate.Thus,the capture of chemoresistance became a hotspot in cancer research.A promising approach to overcome chemoresistance is drug screening from natural products.Previously,NK007 was synthesized as a tylophorine analog,which helped in mitigating paclitaxel resistance in human ovarian cancer.Moreover,NK007 and PTX synergically enhance their anti-tumor effect.These results suggested that NK007 effectively overcome paclitaxel resistance in human ovarian cancer,which urged us to study molecular mechanism further.Objective: This study focus on molecular mechanism in overcoming paclitaxel resistance with NK007 in human ovarian cancer;providing cellular and molecular biological evidence of NK007 as p38 MAPK agonist;exploring the interaction between hexokinase and p38 MAPK with NK007.Methods: WST-8 and clone formation assay were used to access the anti-proliferative effect of NK007 in ovarian cancer cell lines;FACS was used to detect the cell apoptosis,cell cycle arrest and cancer stem cell markers;Western blot was performed to detect the protein levels;Gene expression level in ovarian cancer was test by RNA-Seq;DARTS and molecular docking were performed to screen the targets of NK007;Ubiquitination levels of HK2 and the interaction of HK2-p38 MAPK were detected by Co-IP;The effect of NK007 on mitochondrial function and glycolysis was analyzed using the XF96 Cell Mito Stress Test kit.Results: 1.NK007 selectively inhibits cell viability in A2780 and A2780(Taxol)cells.2.NK007 inhibits proliferation and colony formation of A2780 and A2780(Taxol)cells.3.NK007 inhibits proliferation and migration of A2780 and A2780(Taxol)cells but does not affect cell apoptosis.4.NK007 and PTX synergically enhance their anti-tumor effect in A2780 and A2780(Taxol)cells.5.NK007 overcomes paclitaxel(PTX)resistance of A2780 independent of My D88,CDCP-1 and CD44 expression level.6.NK007 induces G1/S cycle arrest by p38-mediated G1 restriction point.7.p38 MAPK inhibitor or si RNA rescued NK007-mediated cycle arrest and growth suppression.8.The DARTS method for drug target identification.9.Identification of NK007 as a potent p38 MAPK agonist by molecular docking.10.NK007 downloaded the expression of HK2 protein and inhibited cell autophagy.11.NK007 promoted the K48-linked ubiquitylation of HK2.12.NK007 restrained glucose uptake.13.NK007 caused a remarkable reduction of oxygen consumption.14.NK007 significantly decreases glycolysis.Conclusions: Our findings highlighted that NK007,a(±)-tylophorine malate isolated from the Asclepiadaceae family,overcomes PTX resistance through two important molecular mechanisms: i)up-regulation of p-p38 MAPK triggers G1/S cycle arrest and ii)HK2 degradation results in metabolic reprogramming in human ovarian cancer.Therefore,cancer cell viability,including cell proliferation,clonality,and migration,is notably inhibited by NK007,which makes it a promising therapeutic agent to treat patients with PTX-resistant ovarian cancer.