April 2018
Mohammad Reza Sam, Mohammad Tavakoli-Mehr, and Reza Safaralizadeh

 

Abstract

Background

The presence of chemotherapy-resistant colorectal cancer stem cells (CCSCs) with KRAS mutation is thought to be one of the primary causes for treatment failure in colorectal cancer (CRC). P53, survivin, and microRNA-16-1 are challenging targets for anticancer drugs which are associated with chemoresistance in CRC. Yet, no p53-, survivin-, and microRNA-16-1-modulating drug with low toxicity but high efficacy against KRAS-mutant CCSCs have been approved for clinical application in CRC. Here, we investigated whether in vitro concentrations of DHA equal to human plasma levels, are able to modulate, Wt-p53, survivin, and microRNA-16-1 in CRC cells with stem cell-like properties.

 

Methods

Wt-p53/KRAS-mutant CRC cells (HCT-116) with stem cell-like properties were treated with 100-, 150- and 200-μM/L DHA, after which cell number, viability, growth inhibition, Wt-p53, survivin and microRNA-16-1 expression, caspase-3 activation and apoptotic-rate were evaluated by different cellular and molecular techniques.

 

Results

After 24-, 48-, and 72-h treatments with 100- to 200-μM/L DHA, growth inhibition- rates were measured to be 54.7% to 59.7%, 73.% to 75.8%, and 63.3% to 97.7%, respectively. Treatment for 48 h with indicated DHA concentrations decreased cell number and viability. In addition, we observed a decrease in both the transcript and protein levels of survivin followed by 1.3- to 1.7- and 1.1- to 4.7-fold increases in the Wt-p53 accumulation and caspase-3 activation levels respectively. Treatment with 100 and 150 μM/L DHA increased microRNA-16-1 expression levels by 1.3- to 1.7-fold and enhanced the microRNA-16-1/survivin mRNA, p53/survivin, and caspase-3/survivin protein ratios by 1.7- to 1.8-, 1.3- to 2.6-, and 1.3- to 2-fold increases respectively. A decrease in the number of live cells and an increase in the number of apoptotic cells were also observed with increasing DHA concentrations.

 

Conclusion

Wt-p53, survivin, and microRNA-16-1 appear to be promising molecular targets of DHA. Thus, DHA might represent an attractive anti-tumor agent directed against KRAS-mutant CCSCs.

Keywords: Colorectal cancer stem cells (CCSCs), Colorectal cancer (CRC), Survivin, P53, microRNA-16-1, Docosahexaenoic acid (DHA), Apoptosis


Background

Colorectal cancer (CRC) is one of the most diagnosed malignancies world-wide with high mortality rate [1]. Chemo- and radiotherapy have side effects and are accompanied by local or systemic toxicities. These therapies ultimately lead to the multidrug resistance and tumor relapse in CRC patients [2]. Recent studies have shown that this may be due, at least in part, to the presence of chemotherapy-resistant colorectal cancer stem cells (CCSCs) [3]. Cancer stem cells are a subpopulation of tumor-initiating cells within a tumor that are able to self-renew and therefore to drive tumor formation. Due to the activation of multiple resistance mechanisms in these cells, cancer stem cells display high resistance to the chemo- and radiotherapy [4–6]. Therefore, new therapeutic approaches, which target CCSCs, are urgently required.

Multiple mediators such as proto-oncogenes, tumor-suppressor genes, and microRNAs contribute to CRC disease pathogenesis. Thus, simultaneously targeting multiple cancer-related genes in CCSCs with a therapeutic compound may provide a better efficacy than activation or inhibition of a single target gene.

One of the key genes regulating cell death is survivin, which belongs to the family of inhibitors of apoptosis proteins. Survivin functions as an oncogene in cancer cells [7] and is overexpressed in CCSCs but rarely in normal cells. Overexpression of survivin has been identified as a negative prognostic factor in CRC and to be implicated in resistance to apoptosis induction by chemotherapeutic compounds [8, 9].

P53 is a key tumor-suppressor protein which controls the cellular response to stress signals through the induction of apoptosis or cell-cycle arrest and thereby prevents neoplastic progression [10, 11]. Mutation of p53 commonly occurs in nearly half of all human malignancies and contributes to tumor progression and development [11, 12]. P53 has also been shown to be associated with the epithelial-mesenchymal transition (EMT), a process which stimulates epithelial cells to acquire the invasive and metastatic properties of mesenchymal cells [13] and has thus been demonstrated to play a critical role in promoting metastasis of epithelia tumors [14] including CRC [15, 16].

MicroRNA-16 functions as a tumor-suppressor microRNA and is downregulated in colon cancer [17]. MicroRNA-16 targets several cell cycle regulators, including different cyclins and cyclin-dependent kinases [18–26]. Overexpression of microRNA-16 induces apoptosis in CRC cells through the intrinsic apoptosis pathway [27]. Therefore, targeting Wt-p53, survivin, and miRNA-16-1 alone or together in CCSCs by a compound may provide lower toxicity to normal stem cells, as well as its differentiated progeny, and may open up avenues to new therapeutic strategies for CRC-directed therapy.

Several studies have shown an inverse relationship between CRC risk and omega-3 polyunsaturated fatty acids (PUFAs) consumption, suggesting a protective role of these PUFAs against the development of CRC [28]. Additional studies have shown that different steps of the tumorigenic process can be influenced by PUFAs [29–33] with a protective role on normal cells and tissues during ionizing radiation treatment [34]. In this context, It has been shown that consumption of 2.4 g and 4.8 g/day of omega-3 PUFAs yields 138.3 and 205.2 μM/L DHA in human plasma, respectively [35, 36]. With reference to the above-mentioned studies, it is possible that, fish-oil-derived DHA at the physiologic doses could target CCSCs and reduces the risk of colorectal cancer incidence in individuals consuming fish. With this in mind, we provided DHA at concentrations equal to those detected in human plasma and treated colorectal cancer stem-like cells harboring KRAS mutation.

KRAS mutations are found in approximately 40% of human CRCs [37] are able to activate CCSCs. In the oncogenic signal networks of CRC, mutated KRAS has been shown to serve many functions beyond maintaining cellular proliferation, stemness and growth factor-independent growth and contributes to colorectal tumorigenesis, metastasis and resistance to therapy [38, 39].

In this study, we aimed to determine whether in vitro concentrations of DHA (100-, 150- and 200 μM/L) equal to the levels of DHA achievable in the human body following supplementation of the diet with different amounts of omega-3 PUFAs/day are able to modulate Wt-p53, survivin, and miRNA-16-1 expression in Wt-p53/KRAS-mutant HCT-116 cells with stem cell-like properties that shows early stages of tumor initiation and development [40–43].

Discussion

Several studies have identified colorectal cancer stem-like cells that are more resistant to cancer treatments due to the multiple resistant mechanisms existing in these cells [3]. CCSCs are the crucial component leading to tumor recurrence [3]. Therefore, CCSC-targeted therapies may have the benefit of increased efficacy. In this regard, efforts to develop drugs targeting KRAS-mutant CCSCs remain challenging, as mutated KRAS has been shown to serve many functions, such as involvement in colorectal tumorigenesis, metastasis, and resistance to therapy.

In this study, we investigated the effects of DHA on HCT-116 cells that serve as a model for CCSCs [40, 42, 43]. This cell line is known to be highly aggressive cells with little or no capacity to differentiate and contains a high proportion of CCSCs that have lost the capacity to differentiate [42, 43]. In view of the fact that the HCT-116 cell line consist almost exclusively of CCSCs cannot be separated into different types of colony-forming cells, nor into different categories with respect to the ability to form tumors in NOD/SCID mice [42, 43], we did not isolate the cell fraction characterized of putative colorectal cancer stem cells for our experiments.

In our study, we found that DHA is a potent agent in decreasing cell number and cell proliferation-rate with induction of apoptosis in KRAS-mutant HCT-116 cells. These data raise the possibility of therapeutic application of this compound against CCSCs carrying KRAS mutations.

Expression of the Wt-p53 is powerfully activated by DHA in HCT-116 cells resulting in accumulation of the Wt-p53 protein. To evaluate, the activity of induced Wt-p53 accumulation, evaluation of downstream p53-target genes expression including survivin and microRNA-16 [27, 45–47] is highly appreciated and may provide indirect documents about the Wt-p53 gain of function in DHA-treated HCT-116 cells. In this regard, it has been shown that survivin is a p53 target gene. The survivin gene promoter contains a p53 response element and increased expression of p53 represses survivin promoter activity, resulting in decreased survivin protein expression [45–47]. Based on our data, low survivin protein levels combined with high levels of Wt-p53 protein accumulation were associated with higher caspase-3 activation levels and apoptosis, suggesting an attractive effect of DHA on the Wt-p53 gain of function and a role for p53 in DHA-mediated apoptosis in HCT-116 cells.

Dysregulation of the Wnt, Notch, Hedgehog, and/or TGF-β signaling pathways which are involved in proliferation and maintenance of CCSCs leads to the development of CRC [48]. These embryonic pathways can interact with other cellular signaling pathways, such as those involving NF-κB, RAF/MAPK/ERK, PI3K/Akt/mTOR, and Wnt/β-catenin [49] of which survivin is a direct downstream target [50–52]. In the present study, we found that DHA decreased survivin expression at both the transcript and protein levels and this followed by accumulation of Wt-p53, caspase-3 activation, and apoptosis, suggesting a role of survivin in p53-dependent apoptosis in HCT-116 cells. In line with the present results, previous reports showed that DHA decreases survivin expression in the LS174T human CRC cell line with stem cell-like properties [53] and induces Wt-p53 accumulation in the acute lymphoblastic leukemia (ALL) Molt-4 cell line [54]. However, these interesting results were mainly descriptive without any mechanistic approach. Further experiments are required to highlight these issues in this regard.

MicroRNA-16-1 is located at chromosome 13q14 [55] and acts as a tumor-suppressor microRNA in different kinds of cancers including CRC [27]. Our findings showed that DHA induces a significant increase in the expression of microRNA-16-1 in a dose-dependent manner followed by apoptosis. Consistent with our study, it has been shown that microRNA-16 overexpression inhibits cell proliferation and induces apoptosis in CRC [27].

To gain an insight about, the functional role of increased microRNA-16 in DHA-treated HCT-116 cells, evaluation of microRNA-16-target mRNA expression is highly valued. Survivin mRNA is a direct target of microRNA-16 in CRC cells [27], and it has been shown that overexpression of microRNA-16 decreased survivin expression in CRC [27]. With this in mind, in our study, one of the mechanisms contributing to downregulation of survivin at both mRNA and protein levels may be related to the interaction of microRNA-16-1 with survivin mRNA in HCT-116 cells.

Wt-p53 enhances the maturation of different microRNAs including microRNA-16 [56, 57]. In this regard, Ma et al. showed that overexpression of p53 causes a significant increase in the levels of microRNA-16 in HCT-116 cells [27] which was consistent with our work. As microRNA-16 is a target of p53 [27], increased expression levels of microRNA-16 may imply the activity of increased p53 accumulation level in DHA-treated cells.

In our study, DHA at concentrations of 100 and 150 μM/L induced significant apoptosis. But, at both concentrations, no significant increase (a slight increase) in caspase-3 activation level was observed. This agrees with previous study showing that DHA-mediated apoptosis can occur in a caspase 3-independent fashion [58].

It is interesting to note that DHA at concentrations of 100 to 200 μM/L equal to plasma levels achievable in the human body following supplementation of the diet with different amounts of ω-3 PUFAs/day [36] modulated Wt-p53, survivin, and miRNA-16-1 and killed CRC-initiating cells with stem cell-like properties through induction of apoptosis and not necrosis suggesting that DHA at concentrations of 100 to 200 μM/L activates apoptosis pathways and not necrosis pathways in KRAS-mutant HCT-116 cells. These results were obtained using DHA concentrations which were below the maximum tolerated dose of omega-3 PUFAs reported in humans [36] demonstrating that DHA could be used as a safe and nontoxic compound. In this regard, numerous studies have reported that DHA has cytotoxic effect toward different kinds of cancer cells [59, 60] with no toxicity on normal cells including peripheral blood mononuclear cells (PBMCs) [61].

It has been shown that DHA induced apoptosis in human colon carcinoma cells COLO 205, carrying Wt-p53 and WiDr colon carcinoma cells containing mutated p53 [32]. These findings argue that DHA exerts both p53-dependent and p53-independent apoptotic effects in colorectal cancer cells [32]. Based on our data and that of other researchers, DHA is a safe and well-tolerated natural compound at concentrations equal to human plasma levels with no detrimental effects on normal hematopoiesis [59] and may therefore find a therapeutic application in CRC patients with Wt- or mutant p53.

In our experiments, the miRNA-16-1/survivin mRNA, p53/survivin protein, and caspase-3/survivin protein ratios were significantly increased as compared to untreated cells and were associated with apoptosis in HCT-116 cells. Evaluation of these ratios would be important and may find a therapeutic index particularly when DHA as a safe adjuvant is used for treatment of CRC with KRAS mutation.


Conclusion

P53, survivin, and microRNA-16-1 are challenging targets for anticancer drugs in CRC which are associated with chemoresistance. Yet, no p53-, survivin-, and microRNA-16-1-modulating drug with low toxicity and high efficacy has been approved for clinical application in CRC. Our observations provide the first evidence that DHA is a valuable safe compound that modulates Wt-p53, survivin, and microRNA-16-1 in CRC-initiating cells with stem cell-like properties and this compound with high pro-apoptotic capacity represents an attractive anti-tumor agent against CCSCs with KRAS mutation.

 

Acknowledgements

The authors acknowledge Dr. Talya Dayton from Hubrecht Institute for her valuable suggestions with respect to the manuscript.