低水平的反应性氧化物质（ROS）通过溶酶体诱导的自噬诱导促进细胞存活。葡萄糖压力所引起的酸中毒、低氧、ROS升高，可上调与肿瘤干细胞和多药物耐受性相关的标记物。另外，有人认为溶酶体上调是ROS诱导应激下细胞存活的重要指标之一。研究支持细胞溶酶体-TFEB-Ca2+级联刺激在诱导化疗耐受性和癌细胞存活中具有重要作用。为了观察卡恩菲醇和维拉帕米联合应用对调节化学侵袭、肿瘤干性、酸中毒以及溶酶体上调途径标记物的影响，我们在低和高葡萄糖条件下进行了实验。基于我们之前的观察和以前的研究，我们假设卡恩菲醇与维拉帕米的联合应用可以减弱化学侵袭、肿瘤干性和酸中毒标记物的表达以及溶酶体-TFEB-Ca2+途径的影响，这些在化疗耐受性中具有不可或缺的关联和作用。在改变的葡萄糖条件下，使用KV处理外体模型，测量候选基因的RNA和蛋白质表达、ROS产生和Ca2+浓度。同时采用计算方法推断药物联合作用的机制。本研究采用PCR、IHC、Western blotting和分子对接等方法。我们的候选药物KV过度产生的ROS抑制了化疗耐受性和肿瘤酸中毒标记物的表达，并导致溶酶体破坏以及Ca2+释放减少，在低葡萄糖条件下降低了TFEB的表达。在高葡萄糖条件下观察到异常的结果。我们还观察到KV促进ROS水平的过度产生，从而通过上调LC3-II和p62在低葡萄糖条件下诱导自噬介导的细胞死亡。外体研究与计算模拟的研究结果相一致。我们的外体和计算模拟研究表明，我们的候选药物联合使用KV可以有效地靶向调节化疗耐受性的多个途径，这些途径之前未曾在相同的实验设置下研究，因此可以用于治疗目的。 版权所有©2023 Elsevier GmbH。

Reactive oxygen species (ROS) at low level promotes cell survival through lysosome induced autophagy induction. Glucose stress induced acidosis, hypoxia, ROS, upregulates markers related to cancer stemness and multidrug resistance. Also, lysosomal upregulation is proposed to be one of the important indicators of cell survival under ROS induced stress. Studies supported that, stimulation of Lysosome-TFEB-Ca2+ cascade has important role in induction of chemoresistance and survival of cancerous cells.To observe the effect of synergistic drug combination, Kaempferol and Verapamil on markers regulating chemoevasion, tumor stemness & acidosis as well as lysosome upregulation pathways, under low as well as high glucose conditions.Based on our earlier observation as well as previous reports, we hypothesized, our drug combination Kaempferol with Verapamil could attenuate markers related to chemoevasion, tumor stemness & acidosis as well as lysosome-TFEB-Ca2+ pathway, all of which have indispensable association and role in chemoresistance.RNA and protein expression of candidate genes, along with ROS production and Ca2+ concentrations were measured in ex vivo models in altered glucose conditions upon treatment with KV. Also, computational approaches were utilized to hypothesize the mechanism of action of the drug combination. PCR, IHC, western blotting and molecular docking approaches were used in this study.The overproduction of ROS by our candidate drugs KV, downregulated the chemoresistance and tumor acidosis markers along with ATP1B1 and resulted in lysosomal disruption with reduction of Ca2+ release, diminishing TFEB expression under low glucose condition. An anomalous outcome was observed in high glucose conditions. We also observed KV promoted the overproduction of ROS levels thereby inducing autophagy-mediated cell death through the upregulation of LC3-II and p62 in low glucose conditions. The ex vivo studies also corroborate with in silico study that exhibited the parallel outcome.Our ex-vivo and in-silico studies revealed that our candidate drug combination KV, could effectively target several pathways regulating chemoresistance, that were not hitherto studied in the same experimental setup and thus may be endorsed for therapeutic purposes.Copyright © 2023 Elsevier GmbH. All rights reserved.