Panoply™ Human CLEC12A Over-expressing Stable Cell Line

Increased inflammatory responses activate Toll-like receptors (TLRs), promoting tumor progression. TLRs, in turn, are inhibited by C-type lectin-like receptors (CTLRs), such as CLEC12A. In hematopoietic tumors, CLEC12A primarily inhibits TLRs and regulates inflammatory responses through NF-κB signaling. Here, researchers used artemisinin (ART) to modulate CLEC12A to determine the fate of tumor progression. Artemisinin is an FDA-approved antimalarial drug known for its anticancer and immunomodulatory properties with minimal side effects on normal cells. Studies have shown that ART does not alter the physiology of normal mice, while in contrast, in tumor-bearing mice, ART leads to tumor regression. Furthermore, ART reduces CLEC12A expression. As expected, TLR4 expression increases, but surprisingly, NF-κB (RelA) and JNK/pJNK expression decreases, accompanied by reduced inflammation, decreased autophagy, and increased apoptosis. All of these observations were reversed by overexpression of CLEC12A in MDA-MB-231 and 4T1 cells. However, TLR4 inhibition did not alter the expression of CLEC12A, NF-κB, or apoptosis markers. The effects of ART on cancer stem cell (CSC) populations follow similar trends as those seen in cancer cells. The interaction and regulation of CLEC12A with ART induces tumor cell death and eliminates CSCs, demonstrating that ART represents a more comprehensive cancer treatment approach with a reduced risk of relapse. Therefore, ART may be repurposed as an effective cancer therapy in the future.

Immunoblotting revealed increased expression of the anti-apoptotic protein (Bcl-2) and decreased expression of pro-apoptotic markers in CLEC12A-overexpressing cells compared with untransfected and empty vector-transfected cells. However, ART treatment failed to increase the expression of these proteins in CLEC12A-overexpressing cells compared with untransfected and empty vector-transfected cells. Similar results were observed in both mouse and human cell lines (Figure 1A). To further confirm the immunoblot data, Annexin-FITC and PI staining were performed to estimate the number of apoptotic cells by flow cytometry. An increase in the percentage of apoptotic cells in response to ART treatment was observed in both cell lines (0.2% to 11% in 4T1 cells and 0.5% to 22% in MDA-MB-231 cells). However, ART treatment did not significantly increase the percentage of apoptotic cells in CLEC12A-overexpressing cells (Figure 1B). To test whether ART induces tumor cell apoptosis by downregulating CLEC12A and autophagy, cells were first treated with the autophagy inhibitor 3-methyladenine (3-MA) before ART treatment of normal cells and CLEC12A-overexpressing cells. Western blot analysis revealed that upon 3-MA treatment of CLEC12A overexpressed cells, the levels of anti-apoptotic protein (Bcl-2) was significantly higher in both cell lines compared to non-transfected cells. Following ART treatment, Bcl-2 expression remained significantly elevated in CLEC12A-overexpressing cells compared to ART-treated untransfected cells. Furthermore, the expression of pro-apoptotic markers remained significantly lower after ART treatment compared to ART-treated untransfected cells (Figure 1C).

Figure 1. CLEC12A overexpression restricts the ability of artemisinin to induce apoptosis. (Chatterjee R, et al., 2023)