Colorectal cancer is one of the prevalent widespread forms of cancer globally. Acquired biological properties and functional capabilities of colon cancer cells have important hallmarks such as immortality, sustaining cell proliferative signaling, and stimulating angiogenesis and metastasis. Also, the tumor microenvironment is a fundamental factor in the initiation, progression, metastasis, and therapy response of colon cancer cells . Additionally, the metastatic properties of colon carcinoma or metastasis of other types of cancer cells into the colon are other issues that need to be investigated .
According to CDC data, the 5- year survival percentage for colorectal cancer cases is 63.8%. Besides surgical intervention and radiotherapy, chemotherapy is the most common cancer treatment method . However, chemoresistance and the side effects of chemotherapeutic drugs have led to the search for new agents with fewer side reactions in cancer treatment. Polyphenols are natural compounds that are used for chemoprevention and alternative approaches to decrease the mortality rate in CRC. Various researches have demonstrated that polyphenols can inhibit the overall carcinogenesis process by stimulating apoptosis through several mechanisms. Quercetin, which belongs to the natural polyphenols group, is the most studied flavonoid against CRC. Quercetin is commonly found in onion, apple, strawberry, broccoli, tea, and red wine, which are frequently included in our diet . In recent years, due to its effects on biosignalization and carcinogenesis, quercetin has been frequently used in anti-cancer research .
Recent experimental research have demonstrated that quercetin inhibits cancer cell proliferation and viability based on incubation time and dosage. Rafiq et al., found that the application of B16F10 melanoma cell line with 5µM quercetin for 24 hours affected cell viability . Also, Zhang et al., found that the application of Caco- 2 and SW- 620 cell lines with 20 µM quercetin application for 24 hours had an effect on cell viability . According to the study of Han et al., Caco- 2 cell viability was inhibited as a result of 20 µM quercetin application for 24 hours . On the basis of our previous report, the affective concentration was found to be 25 µg/ml for 48 hours in two of the cell lines Colo 320 and Colo 741 .
Exosomes are nano-sized vesicles and a crucial aspect of the tumor- microenvironment. Exosomes and exosom-derived miRNAs or proteins that are released by tumor cells contribute to the tumor micro-environment and may play an essential role in CRC progression. Therefore, there has been increased attention to cargo molecules that are found in exosomes as potential tumor markers and treatment targets for colorectal cancer . They can transfer miRNAs, proteins, mRNAs, and DNA fragments from donor cells to distant recipient cells . Recently, accumulating evidence shows that differentially expressed proteins and miRNAs in exosomes are latent novel biomarkers for CRC confirmation. Additionally, studies have demonstrated that different miRNAs and protein content in the blood exosomes of CRC patients may be beneficial prognostic predictors and would support the determination of innovative therapeutic strategies [34, 35]. Up to the present time, no study has investigated the effects of quercetin with respect to the expression of exosome marker proteins and miRNA biogenesis related proteins and exosomal miRNA levels in Colo 320 and Colo 741 tumor cells.
In our study, we reported that the immunoreactivity of CD9 has statistically significant increase in quercetin applied Colo 741 cells when compared to the control group. Also, the immunostaining intensity of CD9 was significantly higher in Colo 741 cells than Colo 320 cells after administration of quercetin. On the other hand, the immunoreactivity of CD63 was increased in Colo 320 primary colon cancer cell line and decreased in Colo 741 metastatic colon cancer line, after the application of quercetin. The immunostaining intensity of CD63 has statistical significant elevation in quercetin applied Colo 320 cells when compared with quercetin applied Colo 741 cells. It can therefore be finalized that Colo 320 and Colo 741 colon cancer cells secreted exosomes in response to quercetin administration and to use of exosomes for colon carcinoma identification, both CD9 and CD63 should be used because of diverse exosomal surface proteins secretion.
miRNAs are non-coding RNAs that regulate a large number of biological processes by inhibiting target gene expression. Functional studies have investigated that miRNAs have pivotal functional roles in CRC carcinogenesis and prognosis. Furthermore, emerging evidence indicates that miRNAs are robust biomarkers for the diagnosis and surveillance of CRC patients . Current evidence indicates that miRNAs biogenesis and related proteins such as Dicer and Ago2 have effects on the development of several cancers. Chiosea et al., showed that pre-cancerous lesions and invasive lung adenocarcinomas were related to Dicer gene locus deletion . The molecular mechanisms that regulate Dicer-related carcinogenesis in CRC are still recondite. Faber et al., showed that Dicer protein overexpression could serve as a poor prognosticator in CRC . Nonetheless, previous studies have documented that Dicer mRNA levels were not significantly correlated with patient survival [14, 37]. On the other hand, Iliou et al., reported that Dicer defects were linked to the colorectal cancer cells subpopulations generation with upregulated stem cell markers . In light of these well-documented reports, we establish that the immunoreactivity of Dicer was increased in both quercetins applied Colo 320 and Colo 741 cells lines when compared with their control groups, but the elevations have no statistical significance. Moreover, the immunoreactivity of Dicer significantly increased in quercetin applied Colo 741 cells in comparison to quercetin applied Colo 320 cells. These data lead to the hypothesis that quercetin could have protective effects by the upregulation of tumor suppressive miRNA by increasing Dicer expression in colorectal cancer.
Ago2 is a key regulator of miRNAs in target mRNAs degradation through its catalytic activity in processes of gene silencing. The Ago2 protein has been found to be over-expressed in colon cancer, ovarian carcinoma, gastric carcinoma and colorectal carcinoma [39, 40, 41, 14]. Additionally, it has been shown that Ago2 over- expression was related to cancer aspects, including tumor cell proliferation, growth, and overall cancer patient survival . However, the expression level of Dicer can be different from Ago2 expression level. It was found that the upregulated Dicer mRNA expression levels were not positively correlated with Ago2 mRNA expression levels . Our results revealed that Ago2 immunoreactivity was decreased in quercetin applied Colo 320 and Colo 741 cells when compared with control groups, the decreases have not any statistical significance in both cell lines. Therefore, it may be regarded that the incubation time needs to be lengthened in order to detect a statistically significant decrease in Ago2 immunoreactivity after quercetin application in two cell lines Colo 320 and Colo 741.
Recent studies have reported that quercetin can elicit obvious endoplasmic reticulum stress in a variety of tumor cells [44, 45]. The different type of cellular stress leads to translation downregulation in cells, which is thought to save energy and encourage survival. The best-characterized downregulation mechanism for the translation regulation is the phosphorylation of eukaryotic initiation factor eIF2α in eukaryotic cells. Also, translational attenuation can stimulate apoptosis and autophagy in cells . According to our experimental results, the staining intensity of eIf2α was decreased in all two Colo 320 and Colo 741 colon cancer cell lines after quercetin application; however, the decrease was only significant in the Colo 741 colon cancer cell line. In our very recent publication, we found that quercetin triggered apoptosis in Colo 320 and Colo 741 cells, which agrees with the findings of this study . The results indicated that quercetin may have protective effects by downregulating eIF2α expression and promoting apoptosis in metastatic colon cancer cells.
miRNAs are single stand non-coding RNAs that suppress mRNA translation and stimulate mRNA degradation. Experimental studies have documented that irregular miRNA levels in colorectal cancer may regulate tumorigenesis . Exosomes are known as vehicles for miRNAs and are miRNA biomarker sources in bodily fluids . According to our data, in comparison between control groups and quercetin application groups, total exosomal miRNA concentrations were increased in two primary and metastatic cell lines after quercetin application, but the elevations have no statistical significance.