Enhancement of 3-MA in Paclitaxel Treatment of MDA-MB-231 Tumor-Bearing Nude Mice and Its Mechanisms

Abstract

Triple-negative breast cancer (TNBC) poses significant challenges due to its high aggressiveness, poor prognosis, and the lack of effective targeted therapies. Paclitaxel (PTX) is a chemotherapeutic agent commonly used in the treatment of TNBC; however, its efficacy is often compromised by drug resistance mediated by autophagy. This study investigated the synergistic effects of the autophagy inhibitor 3-methyladenine (3-MA) and PTX in a TNBC nude mouse model. Monitoring tumor volume and employing HE staining, immunofluorescence, and transmission electron microscopy revealed that PTX monotherapy induced tumor autophagy, characterized by the accumulation of LC3B/VPS34 proteins and an increase in autophagosomes. However, the co-administration of 3-MA reversed this process, significantly decreasing the tumor growth rate. Immunofluorescence and qPCR demonstrated that the combination group had fewer Ki-67-positive cells and more Caspase-3-positive cells, along with upregulated expression of autophagy-related genes and Caspase-family apoptosis genes. Consequently, this study suggests that inhibiting autophagy with 3-MA disrupts the autophagy-mediated protective mechanism of tumor cells, promoting the activation of apoptotic signals and enhancing the antitumor activity of PTX. These findings may offer new molecular mechanistic insights and potential therapeutic strategies for overcoming PTX resistance in TNBC.

Keywords: 3-MA; MDA-MB-231; autophagy; paclitaxel; triple-negative breast cancer.

Figure 1

Comparison of tumor growth and body weight curve in MDA-MB-231 cell-transplanted and tumor-mass-transplanted nude mice. (A) Statistics on tumor volume in MDA-MB-231 cell-transplanted and mass-transplanted group. ** p < 0.01 indicates a significant difference; (B) Changes in body weight of normal control group, cell-transplanted group and mass-transplanted group; (C) HE staining images of adjacent muscle tissue and tumor tissues; (D) Immunofluorescence images of Ki-67 in muscle and tumor tissues.

Figure 2

Effect of tumor volume on the therapeutic efficacy of paclitaxel in tumor−bearing nude mice. (A) Tumor volume measurements of mass-transplanted tumor models over 16 days; (B) Tumor changes on days 0, 10, and 20 during treatment in tumor-bearing nude mice. (C) Tumor volume statistics of nude mice treated with PTX, 3-MA, and PTX + 3-MA for 28 days (n = 10); (D) Subcutaneous tumor mass dissection in tumor-bearing nude mice.

Figure 3

HE staining of tumor masses and organ tissues in MDA-MB-231 tumor-bearing nude mice treated with PTX and 3-MA. The morphology of the tumor, heart, liver, spleen, lung, and kidney tissues in the Control, PTX, 3-MA, and PTX + 3-MA groups were observed through HE staining. Scale bar = 200 μm.

Figure 4

Transmission electron microscopy images of tumor tissues in MDA-MB-231 tumor-bearing nude mice treated with PTX and 3-MA. The tumor tissues autophagosomes in the control, PTX, 3-MA, and PTX + 3-MA groups were observed through transmission electron microscope. Scale bar = 2 μm. The structure indicated by the red arrow is an autophagosome.

Figure 5

The effects of PTX and 3-MA combination on the expression of Ki-67, LC3B, VPS34, and Caspase-3 in tumor tissues. The expression of Ki-67, LC3B, VPS34, and Caspase-3 in the tumor tissues for the control, PTX, 3-MA, and PTX + 3-MA groups were detected by immunofluorescence. Scale bar = 100 μm.

Figure 6

Expression of related genes in MDA-MB-231 tumor-bearing nude mice treated with PTX and 3-MA combination. (A) mRNA expression levels of ATG10 and FAS; (B) mRNA expression levels of DRAM2, GSTK1, and MYCBP; (C) mRNA expression levels of STK4 and STK38; (D) mRNA expression levels of VPS29 and VPS50; (E) mRNA expression levels of CASP1, CASP2, and CASP8. GAPDH was used as the internal reference; n = 3, * p < 0.05 or ** p < 0.01 indicates a significant difference. ^ns p > 0.05 indicates no significant differences.