Increased knowledge about CRC pathogenesis has provided
Increased knowledge about CRC pathogenesis has provided new and remarkable treatments beyond the classical options of surgery, radiation and chemotherapy . Despite these advances, chemotherapy remains the most important element of anti-cancer therapy. Chemotherapy, when initiated, is generally effective in minimizing tumor growth; however, its limitations often lead to disease relapse and death. In particular, the failure rate for chemotherapy is high in metastatic cancer, and this failure is mainly caused by chemoresistance . Thus, there is a need to delineate the mechanisms of chemoresistance in tumor cells. Recently, it has been proposed that resistant tumor cells represent a small fraction of phenotypically distinct cells that are unlike most tumor cells. These cells are called cancer-stem-like cells or cancer stem cells (CSCs). It is believed that only CSCs are capable of tumor initiation, proliferation, and metastasis . CSCs are detected in many solid tumors, including CRC [, , ] and are identified either by the expression of specific cell surface markers, such as CD133, CD44, CD24, CD29, CD166, Lgr5, and the colon stem cells marker DCLK1 or by detoxifying enzymes such as aldehyde dehydrogenase 1 (ALDH1). Previously, we demonstrated that ALDH-positive cancer-initiating cells (CICs) impart resistance against Fluorouracil (5-FU) treatment in CRC cell lines . Additionally, we demonstrated the CRC therapeutic potential of the CysLT1R receptor antagonist montelukast (Mo) . The therapeutic benefits of Mo have been reported in other inflammatory disorders such as AZD-9291 mesylate mg  and rheumatoid arthritis . Through our previous research, Mo reduces tumor development in a xenograft model of CRC . In the current study, we investigated whether inhibition of CysLT1R could prevent colon cancer-CSCs and associated deleterious factors that participate in tumor growth.
Materials and methods
Discussion In earlier research, we established that the inflammatory leukotriene LTD4 potentiated tumor growth from CICs, expressing high ALDH . Moreover, its receptor CysLT1R has been implicated in the progression of CRC [6,7]. Based on these studies, we explored whether blocking CysLT1R could be an useful approach in minimizing tumor growth, with a possible inhibitory effect on CSCs, and associated factors. To achieve this objective, we used the CysLT1R antagonist Mo and studied the in vitro and in vivo effects on colony formation, colonospheres and tumor growth in mice, using two cell lines, HT-29 cells and SW-480 cells, that differ significantly in CysLT1R expression level (Fig. 1A and B). Our in vitro results clearly indicate that Mo inhibited LTD4 induced colony formation in both HT-29 and SW-480 cell lines. We also discovered a strong effect in HT-29 cells in response to treatment corresponding to high levels of CysLT1R in these cells. Interestingly, we noticed phenotypic alterations in the colonospheres with Mo treatment, with noticeable disintegration of the core of HT-29 cells and less significantly for SW-480 cells. Similarly, in the xenograft model Mo treatment resulted in a noticeable decrease in HT-29-derived tumor growth and weight, in mice, over time. Keeping in mind the proposed role of CICs in tumor growth and drug resistance [, , ], we focused mainly on factors related to CICs. Mo-treated colonospheres and tumors contained significantly less ALDH1 protein. ALDH1 is regarded as a CSC marker that belongs to a family of cytosolic (and mitochondrial) enzymes that convert aldehydes to carboxylic acid to minimize cytotoxicity . ALDH1 is proposed to impart survival benefits to CSCs by conferring drug resistance [27,28]. However, in our study, ALDH1 levels were decreased by Mo, which could underlie the increased susceptibility of tumor cells to its effects. We also checked whether the effect of Mo was restricted to ALDH1 presence in CSCs, or if it has a general effect on cancer-stemness, by verifying possible alteration in other prominent CCCSC markers like DCLK1, GLI1 and KLF4. Earlier studies reported DCLK1 as a putative normal stem cell marker in the gut , however, a recent study revealed a short form of DCLK1 (due to transcription at an alternative promoter), and it has been shown to be expressed exclusively in intestinal CSCs . DCLK1 positive intestinal-CSCs were shown to retain their capacity to produce tumors in ApcMin/+ mice, suggesting a role in CRC . Furthermore, upregulated levels of transcription factors GLI1 and KLF4 were reported as typically associated with stem cell related Hedgehog and Notch signaling, imparting self-renewal ability to CSCs [32,33]. Down-regulation of ALDH1 and DCLK1 in both colonospheres and tumors in mice upon Mo treatment, backed by a decrease in GLI1 and KLF4 in mice, these results support our assumption that Mo has a wide spectrum of activity.