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Lentivirus vector-mediated Gag-Caspase-8 induces apoptosis and S-phase arrest in triple-negative breast cancer primary cells
- WANG Min, LI Xiping, TAN Jun, QIU Mei, HOU Zeyu, TIAN Ying, LUO Hongying, FAN Chaowen, QI Ling, YU Qi, XIE Wei
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Journal of Shandong University (Health Sciences). 2025, 63(1):
25-34.
doi:10.6040/j.issn.1671-7554.0.2024.1107
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Objective To investigate the effects and mechanisms of Gag-Caspase-8-containing lentivirus-like particles on the proliferation, migration, cell cycle, and apoptosis in primary triple-negative breast cancer(TNBC)cells. Methods Three clinical specimens of human breast cancer were collected, and primary cells were cultured using an improved tissue block culture method. The cultured cells were identified as high-purity triple-negative breast cancer primary cells through HE staining, immunohistochemistry, and immunofluorescence. Lentiviral transduction was employed to construct virus-like particles(VLPs)carrying Gag-Caspase-8. The cells were divided into the PBS group, Gag-VLPs group, and Gag-CASP8-VLPs group. Cell proliferation was assessed by MTT assay, cell migration was evaluated by scratch assay, apoptosis was detected by AO/EB staining and flow cytometry, and the expression of apoptosis-related proteins was examined by Western blotting. Results Clinical tumor specimens were confirmed as breast cancer tissues by HE staining. Immunohistochemistry revealed high expression of CA153 in the primary breast cancer cells, with a purity of approximately 98% based on the proportion of positive cells. Immunofluorescence analysis showed that HER2, ER, and PR were all negatively expressed, confirming the cultured cells as high-purity triple-negative human breast cancer primary cells. Western blotting detected the expression of the lentiviral vector-specific marker P24 in both Gag-VLPs and Gag-CASP8-VLPs, indicating successful packaging of lentivirus-like particles. Upon intervention with Gag-CASP8-VLPs for 24 and 48 hours, compared to PBS and Gag-VLPs controls, inverted microscopy observed cellular shrinkage, reduced size, decreased adherence, and increased floating cells. MTT assays demonstrated significant inhibition of cell growth in the Gag-CASP8-VLPs group(P<0.01), with time-dependent effects. Wound healing assays showed significant inhibition of cell migration(P<0.05). Flow cytometry revealed a significant increase in S-phase cells(P<0.01), indicating cell cycle arrest at the S phase. AO/EB staining and flow cytometry detected induced apoptosis in the Gag-CASP8-VLPs group(P<0.01). Western blotting results indicated significantly increased expressions of Gag-Caspase-8, Pro caspase-8, Active caspase-8, and Caspase-3(P<0.01), demonstrating that lentivirus-mediated Gag-Caspase-8 effectively enters and activates downstream apoptotic executioner Caspase-3 in triple-negative human breast cancer primary cells, leading to their apoptosis. Conclusion Lentivirus-mediated Gag-Caspase-8 can deliver activated Caspase-8 into primary TNBC cells, induce apoptosis through activation of caspase-3, arrest cells at the S phase, and inhibit cell proliferation and migration.
