The Curcumin Analog PAC Is a Potential Solution for the Treatment of Triple-Negative Breast Cancer by Modulating the Gene Expression of DNA Repair Pathways

Abstract

Breast Cancer (BC) is one of the most common and challenging cancers among females worldwide. Conventional treatments for oral cancer rely on the use of radiology and surgery accompanied by chemotherapy. Chemotherapy presents many side effects, and the cells often develop resistance to this chemotherapy. It will be urgent to adopt alternative or complementary treatment strategies that are new and more effective without these negative effects to improve the well-being of patients. A substantial number of epidemiological and experimental studies reported that many compounds are derived from natural products such as curcumin and their analogs, which have a great deal of beneficial anti-BC activity by inducing apoptosis, inhibiting cell proliferation, migration, and metastasis, modulating cancer-related pathways, and sensitizing cells to radiotherapy and chemotherapy. In the present study, we investigated the effect of the curcumin-analog PAC on DNA repair pathways in MCF-7 and MDA-MB-231 human breast-cancer cell lines. These pathways are crucial for genome maintenance and cancer prevention. MCF-7 and MDA-MB-231 cells were exposed to PAC at 10 µM. MTT and LDH assays were conducted to evaluate the effects of PAC on cell proliferation and cytotoxicity. Apoptosis was assessed in breast cancer cell lines using flow cytometry with annexin/Pi assay. The expression of proapoptotic and antiapoptotic genes was determined by RT-PCR to see if PAC is active in programming cell death. Additionally, DNA repair signaling pathways were analyzed by PCR arrays focusing on genes being related and confirmed by quantitative PCR. PAC significantly inhibited breast-cancer cell proliferation in a time-dependent manner, more on MDA-MB-231 triple-negative breast cancer cells. The flow cytometry results showed an increase in apoptotic activity. These data have been established by the gene expression and indicate that PAC-induced apoptosis by an increased Bax and decreased Bcl-2 expression. Moreover, PAC affected multiple genes involved in the DNA repair pathways occurring in both cell lines (MCF-7 and MDA-MB231). In addition, our results suggest that PAC upregulated more than twice 16 genes (ERCC1, ERCC2, PNKP, POLL, MPG, NEIL2, NTHL1, SMUG1, RAD51D, RAD54L, RFC1, TOP3A, XRCC3, XRCC6BP1, FEN1, and TREX1) in MDA-MB-231, 6 genes (ERCC1, LIG1, PNKP, UNG, MPG, and RAD54L) in MCF-7, and 4 genes (ERCC1, PNKP, MPG, and RAD54L) in the two cell lines. In silico analysis of gene-gene interaction shows that there are common genes between MCF-7 and MDA-MB-321 having direct and indirect effects, among them via coexpression, genetic interactions, pathways, predicted and physical interactions, and shared protein domains with predicted associated genes indicating they are more likely to be functionally related. Our data show that PAC increases involvement of multiple genes in a DNA repair pathway, this certainly can open a new perspective in breast-cancer treatment.

Keywords: DNA repair pathway; PAC; breast cancer; curcumin analog.

Figure 1

Effect of PAC on cell morphology in MCF-7 and MDA-MB 231 cells. Cells were treated with DMSO and PAC at a concentration of 10 μM for 24 h. The results were observed under an inverted microscope; magnification, ×20.

Figure 2

Effect of PAC on MCF-7 and MDA-MB 231 cell viability and cell toxicity. (A,C) Cell viability was determined by the MTT assay respectively in MCF-7 and MDA-MB-231 cells. Cell viability was calculated as a ratio (percentage) of the number of viable cells in the experimental wells to that in the control well. Cells were initially seeded at the density of 3 × 10³ per well, the results are expressed as mean ± S.D. (n = 8). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control. (B,D) Cell cytotoxicity respectively in MCF-7 and MDA-MB 231 cells was used by LDH assay. Cells were treated with DMSO or PAC at a concentration of 10 μM for 24 h. The results are expressed as mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus control.

Figure 3

Effects of PAC on cell migration. Migration of MCF-7 and MDA-MB231 cancer cell lines was assayed by the wound-healing assay.

Figure 4

Effect of PAC on breast cancer cell apoptosis by flow-cytometry. The apoptosis rate of cells in each group was detected using flow cytometry by using Annexin/Pi assay for 24 h and 48 of PAC treatment.

Figure 5

mRNA expression of Bax/Bcl-2. MCF-7 and MDA-MB231 Quantitative real-time PCR was performed to determine the expression level of Bcl-2 and Bax genes. GAPDH was used as a housekeeping gene. The results are expressed as mean ± S.D. (n = 4). ** p < 0.005 and *** p < 0.0005 versus control.

Figure 6

Screening using the RT² Profiler PCR Array to determine the effect of PAC on genes involved in the DNA repair pathway. Scatter plots show the upregulation (yellow dots) by more than 2 fold compared with untreated control cells. (A) In MCF-7, six genes were upregulated. (B) In MDA, 16 genes were upregulated. (C) The schematic diagram represents the upregulation of DNA repair genes in MCF-7 and MDA-MB231cells because of treating them with PAC.

Figure 7

mRNA expression of DNA repair genes in MCF-7 and MDA-MB231. Quantitative real-time PCR was performed to determine the expression level of DNA repair genes. GAPDH was used as a housekeeping gene. The results are expressed as mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus control.

Figure 8

Interaction network of all associated genes in human DNA damage. An interaction network of all associated genes was used for analyzing gene–gene interactions. Input genes predict genes with their categorized functional association implied by genomic was determined by GENEMANIA (University of Toronto, Toronto, ON, Canada), was applied to conduct a network analysis of the common genes and to predict related genes and can be installed from ‘App Manager’ in Cytoscape software (Institute of Systems Biology, Seattle, WA, USA).