الفهرس 
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Abstract
Breast cancer (BC) is a heterogeneous disease and is classified into different subtypes using a variety of clinical and pathological features. Based on immunohistochemical features, TNBC considered as one of the most difficult subtypes of breast cancer to treat thus, chemotherapy till now is the only option to treat TNBC. Recently, different studies have indicated that 1,2,4-triazole-based derivatives exhibit notable cytotoxic activity. It was displayed various therapeutic effects including; antimicrobial, antitubercular, anticancer, anticonvulsant, anti-inflammatory and analgesic. Additionally, 5‑mercapto‑1,2,4‑triazole derivatives in specific proved to be effective anticancer agents in various cancer cell subtypes and show more potency compared to their parent derivatives.
Chemoresistance is another obstacle facing treating various types of BC. It primarily mediated by the expression of ATP-binding cassette (ABC) transporters including; (BCRP1/ABCG2). High expression of BCRP1 protein in cancer cells serves as an indication of drug resistance, aggressiveness, and poor prognosis. In this context, inhibiting BCRP1 could offer therapeutic advantages for cancer treatment. Besides, from the vital signaling pathways that regulates oncogenesis, cancer progression, proliferation, apoptosis, metastasis and chemoresistance in BC is the STAT3 signaling. Thus, inhibition of STAT3 may give a potential therapeutic strategy to develop anticancer agents. Among the novel and promising strategies in STAT3 targeting is the inhibition of the gene expression coding for STAT3 using the technology of Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9).
The present study is an attempt to shed more lights on the potential effect of 3-(4-chloro-2-(trifluoromethyl) anilino)-5-mercapto-4H-1,2,4-triazole (TRZ) as a targeted therapy for BCRP1 protein in STAT3 knocked out subtypes of BC. This might explain more about the possible cross talk between STAT3 signaling pathway and the basic properties of the tumorigenic behavior of BC. To approach the objective of the current study, two different subtypes of BC cell lines were utilized; the luminal type a subtype; MCF-7 and the basal-like type B subtype MDA-MB-231. According to treatment, both BC cell lines were divided into: group (1): Untreated wild cells. group (2): DOXO-treated (wild cells treated with IC50 of DOXO for 48hrs). group (3): TRZ-treated (wild cells were treated with IC50 of TRZ for 48hrs). group (4): STAT3-KO (Cells treated with CRISPR/Cas9 to knock-out STAT3 gene). group (5): KO-DOXO (STAT3-KO cells treated with IC50 of DOXO for 48hrs). group (6): KO-TRZ (STAT3-KO cells were treated with IC50 of TRZ for 48hrs).
In silico investigations were carried out using molecular docking was carried out between four triazole derivatives and the BCRP1 protein (PDB ID: 6ETI) as a bioinformatics tool.
In vitro investigations were done for each group of the used cell lines according to the experimental design as the following; Cell proliferation analysis assay was carried out by MTT assay to determine IC50 values for DOXO and TRZ. Knocking-out of STAT3 Gene by CRISPR/Cas9 Complex was done according to the manufacturer’s protocol and the cutting efficiency of the sgRNA was validated through in vitro cutting enzyme efficiency test. Morphological alterations after treatments were observed using inverted microscope. Cell cycle analysis was carried out using flow cytometer. Molecular investigations were carried out at to assess the level of expression of STAT3, BCRP1, MMP2 and VEGF genes as well as BCRP1 protein level.
In silico analysis highlighted TRZ as a suitable candidate for the study based on its physicochemical properties. In terms of IC50 values, TRZ exhibited higher receptivity with IC50 values of 180μM and 62.0μM for MCF-7 and MDA-MB-231 cell lines, respectively. This suggested that TNBC cells might display greater sensitivity to chemotherapeutic drugs compared to NTNBC cells. The study proposed that variations in phenotypic, metabolic, genetic, and molecular profiles between the MCF-7 and MDA-MB-231 cell lines could account for the differing drug sensitivity observed between these subtypes.
The impact of treatment protocols on cell morphology was investigated. In the case of DOXO treatment, both wild MCF-7 and MDA-MB-231 cells displayed rounded detached cells and debris. However, TRZ treatment led to distinct changes: wild MCF-7 cells became small oval detached cells, while wild MDA-MB-231 cells turned triangular with increased detachment. These changes were likely due to alterations in cytoskeletal structure involving actin polymerization and myosin-mediated contractions. In STAT3-KO cells, elongated cell clusters and circular aggregates formed. Additionally, TRZ induced a shift in STAT3-KO cell morphology towards smaller circular cells. Notably, MDA-MB-231 cells exhibited more circular cells, small aggregates, and debris compared to MCF-7 cells, indicating potential chemoresistance induction in MDA-MB-231 cells.
The effect of treatments on the cell cycle was also investigated. For wild MCF-7 cells, both DOXO and TRZ treatments increased cellular arrest in the G0/G1 phase while reducing proliferative cells in S and G2/M phases. Similarly, wild MDA-MB-231 cells showed increased G0/G1 phase arrest and decreased S phase proliferation after DOXO or TRZ treatment. Compared to DOXO, TRZ seemed more promising against different breast cancer types in terms of its anticancer effects. However, mechanism of action requires further exploration. Knocking out the STAT3 gene in both cell lines resulted in cells appearing in the subG1 phase, indicating late-stage apoptosis due to DNA fragmentation. This was associated with reduced arrests in G0/G1, S, and G2/M phases. Inhibiting STAT3 signaling led to decreased cell viability and induced apoptosis across different cancer types. Additionally, treating STAT3-KO cells with TRZ prompted apoptotic cells in the subG1 phase, lowered arrested cells in G0/G1, and increased proliferative cells in S phase compared to TRZ-treated cells. These findings suggest that TRZ’s cytotoxic, antiproliferative, and pro-apoptotic effects are evident in STAT3-KO breast cancer cells.
Molecular investigations showed the change in the studied genes as the following:
STAT3 gene expression: treatment with TRZ showed a significant down regulation in STAT3 gene expression in wild MCF-7 cells. Inversely, in wild MDA-MB-231 cells, TRZ led to a significant up regulation in STAT3 gene expression but still less than that observed after DOXO treatment. This proposed that the anticancer activity of a triazole derivatives maybe through inhibition of STAT3 signaling pathway. The sensitivity of MDA-MB-231 cells against either treatment with DOXO or TRZ was different resulted in differences in the intensity of cellular responses.
BCRP1 gene expression: TRZ caused a significant down regulation in BCRP1 gene expression in wild cells of MCF-7 and MDA-MB-231. TRZ-induced BCRP1 down regulation level was more pronounced in MDA-MB-231 cells. This could be emphasizing that MDA-MB-231 may respond better to chemotherapeutic agents than MCF-7. Consequently, it could be suggested that TRZ may be the drug of choice in treatment of the aggressive TNBC. Knocking out of STAT3 in BC cells increased their chemosensitivity to DOXO which, in turn, resulted in the inhibition of tumor progression. Meanwhile, treatment of BC cells with TRZ resulted in insignificant changes in gene expression of BCRP1 in both cell lines; MCF-7 and MDA-MB-
231. This might be as result of increasing sensitivity of cells to DOXO treatment while TRZ effect was genuinely high enough to induce further changes after knocking out of STAT3 gene. BCRP1 expression: Assessment of BCRP1 protein level was in consistence with the gene expression.
VEGF gene expression: TRZ treatment caused an insignificant down regulation in the gene expression of VEGF in wild MCF-7 but, reached to an extreme significant level in wild MDA-MB-231. Studies showed that different 1,2,4 triazole derivatives can be used as chemotherapeutic agents through their antiproliferative and antiangiogenic properties. Considering the influence of treatments in both cell lines, it could be concluded that treatment with TRZ may be more favorable for target therapy in TNBC than NTNBC. However, in STAT3-KO cells, treatment with TRZ for both cell lines showed a significant down regulation in VEGF gene expression. Meanwhile, the results exhibited no effect on STAT3-KO MDA-MB-231. Considering the influence of TRZ treatment on MDA-MB-231, it could be concluded that treatment TRZ exhibited its maximum effect on VEGF gene expression thus, leaving no more genes to act on.
MMP2 gene expression: Treatment of both wild cell lines with TRZ resulted in downregulation of MMP2 gene expression. These may point out to the antitumor potential of TRZ through metastasis inhibition. However, in STAT3-KO cells, the down regulation in MMP2 gene expression was more severe due to DOXO treatment than that due to TRZ. This might be attributed to the synergistic effect of the co-treatment of DOXO and STAT3 inhibitors in BC in metastasis inhibition via inhibiting MMP2 at the mRNA and protein levels.
from the present study we can conclude that:
1. Molecular docking investigations of four triazole derivatives and the BCRP1 protein were carried out and revealed the anti-carcinogenic potential of TRZ by acting as a selective inhibitor of BCRP1. Besides, in silico analysis showed its physiochemical properties as: good drug-likeness properties, water solubility, lipophilicity properties, high gastrointestinal absorption, good bioavailability and promising safety profile.
2. The present study suggests TRZ as a promising anticancer agent for the treatment of BC especially TNBC.
3. Also, the results may point out to the molecular characteristics of TRZ as antiproliferative, antimetastatic, inhibiting of chemoresistance and antiangiogenic agent.
4. On the other side, regulatory role of STAT3 in oncogenesis, cancer progression, proliferation, apoptosis, metastasis and chemoresistance may reveal the potential of its inhibition or knocking-out to act as a targeted therapy. In this context, the ability to use TRZ as STAT3 inhibiting agent is provided. Also, knocking-out of STAT3 can play a cooperative role with TRZ in treatment of TNBC cell line through down regulation of chemoresistance, angiogenesis, and metastasis.
5. Although the results may provide TRZ as a promising anticancer agent, further in vivo investigations should be taken to confirm the effects of TRZ and to explore its molecular targets and its health impact and safety.