Induction of Apoptosis by PQ1, a Gap Junction Enhancer that Upregulates Connexin 43 and Activates the MAPK Signaling Pathway in Mammary Carcinoma Cells.

Stephanie Shishido,Thu Nguyen

doi:10.3390/ijms17020178

Stephanie Shishido, Thu Nguyen

Open Access

https://doi.org/10.3390/ijms17020178

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Abstract

The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC50 of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38 MAPK, highlighting that the effect of PQ1 on gap junctions as well as cellular survival via a MAPK-dependent pathway.

Highlights

Breast cancer is the most common cancer in women worldwide, and mortality from breast cancer is consistent due to tumor invasion and metastasis [1]
The present study provided the first evidence that PQ1 activates p38 mitogen-activated protein kinase (MAPK) in an aggressive mammary carcinoma cell line
A novel cell line, FMC2u, from the primary tumor with an aggressive, metastatic phenotype was used to evaluate the role of connexin 43 (Cx43) in PQ1-induced cytotoxicity

Summary

Introduction

Breast cancer is the most common cancer in women worldwide, and mortality from breast cancer is consistent due to tumor invasion and metastasis [1]. The use of a cell line derived from an animal with clinical features of human breast cancer can be utilized to screen drugs prior to use in the animal itself. A number of breast cancer cell lines has been established, a limited number of cell lines is available derived from a transgenic mouse model of spontaneous mammary carcinomas. The establishment of such a line would provide another method to study tumor growth and therapeutic effects despite genetic predisposition for cancer formation. A new cell line, female mammary carcinoma cells (FMC2u), was established from the primary tumor tissue resected from a late stage PyVT mouse with an aggressive, metastatic phenotype. The anticancer effects on FMC2u cell growth were determined for gap junction enhancers (PQs)

Results

Discussion

Conclusion