Wardhani Bantari W K, Louisa Melva, Watanabe Yukihide, Setiabudy Rianto, Kato Mitsuyasu
Biomedical Sciences, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia.
Department of Pharmacology, Faculty of Military Pharmacy, Indonesia Defense University, West Java, Indonesia.
Breast Cancer (Dove Med Press). 2021 Sep 21;13:529-538. doi: 10.2147/BCTT.S325429. eCollection 2021.
Epithelial-mesenchymal transition (EMT) and overexpression of drug efflux transporters have been reported to cause doxorubicin resistance. Our previous study indicated that TMEPAI (transmembrane prostate androgen-induced protein) attenuated doxorubicin sensitivity in triple-negative breast cancer cells. However, how TMEPAI contributes to doxorubicin resistance in TNBC remains unclear. Thus, the present study aimed to elucidate the mechanism of TMEPAI in doxorubicin resistance in triple-negative breast cancer cells.
We used BT549, triple-negative cells wild type (WT), and BT549 TMEPAI knock-out. Both cells were treated with TGF-β 2 ng/mL for 24 hours, followed by TGF-β 2 ng/mL and doxorubicin 12.9 nM for another 24 hours. Afterward, the cells were harvested and counted. Cells were further lysed and used for RT-PCR and Western blot analysis. We determined the expression levels of proliferation, apoptosis, EMT markers, and drug efflux transporters. Additionally, we investigated the expressions of PI3K as well as SMAD3 and AKT phosphorylation.
TNBC cells were shown to be less sensitive to doxorubicin in the presence of TMEPAI. TMEPAI was shown to alleviate the mRNA expressions of apoptosis markers: Bax, Bcl2, Caspase-3, and Caspase-9. Our results indicated that the presence of TMEPAI greatly amplifies EMT and increases drug efflux transporter expressions after doxorubicin treatment. Furthermore, our findings demonstrated that TMEPAI reduced the action of doxorubicin in inhibiting SMAD3 phosphorylation. TMEPAI was also shown to modify the effect of doxorubicin by reducing PI3K expressions and Akt phosphorylation in triple-negative breast cancer cells.
Our findings indicate that TMEPAI promotes EMT and drug efflux transporters at least in part by shifting doxorubicin action from SMAD3 phosphorylation reduction to PI3K/AKT inhibition in triple-negative breast cancer cells.
上皮-间质转化（EMT）和药物外排转运蛋白的过表达被报道可导致多柔比星耐药。我们之前的研究表明，跨膜前列腺雄激素诱导蛋白（TMEPAI）会减弱三阴性乳腺癌细胞对多柔比星的敏感性。然而，TMEPAI如何导致三阴性乳腺癌的多柔比星耐药仍不清楚。因此，本研究旨在阐明TMEPAI在三阴性乳腺癌细胞多柔比星耐药中的作用机制。
我们使用了BT549三阴性野生型（WT）细胞和BT549 TMEPAI基因敲除细胞。两种细胞均用2 ng/mL的转化生长因子-β（TGF-β）处理24小时，随后用2 ng/mL的TGF-β和12.9 nM的多柔比星再处理24小时。之后，收集细胞并计数。细胞进一步裂解后用于逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析。我们测定了增殖、凋亡、EMT标志物和药物外排转运蛋白的表达水平。此外，我们研究了磷脂酰肌醇-3激酶（PI3K）以及SMAD3和蛋白激酶B（AKT）磷酸化的表达情况。
在存在TMEPAI的情况下，三阴性乳腺癌细胞对多柔比星的敏感性较低。TMEPAI可减轻凋亡标志物：Bax、Bcl-2、半胱天冬酶-3（Caspase-3）和半胱天冬酶-9（Caspase-9）的mRNA表达。我们的结果表明，TMEPAI的存在极大地增强了EMT，并在多柔比星处理后增加了药物外排转运蛋白的表达。此外，我们的研究结果表明，TMEPAI降低了多柔比星抑制SMAD3磷酸化的作用。TMEPAI还通过降低三阴性乳腺癌细胞中PI3K的表达和Akt磷酸化来改变多柔比星的作用效果。
我们的研究结果表明，在三阴性乳腺癌细胞中，TMEPAI至少部分通过将多柔比星的作用从降低SMAD3磷酸化转变为抑制PI3K/AKT，从而促进EMT和药物外排转运蛋白。
