HT-29, SW620 and MDA-MB-231 cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were maintained in a monolayer culture in DMEM/F12 (Dulbecco's modified Eagle's medium) with 10% fetal bovine serum, 2.5% L-glutamine and 0.5% penicillin/streptomycin.

Morin hydrate (Sigma-Aldrich, St. Louis, MO, USA; catalog no. M4008) and MST-312 (Sigma-Aldrich; catalog no. M3949) was purchased from Sigma-Aldrich Co. Morin was prepared in 50 mM stock solution and MST-312 was in 10 mM stock solution. The working concentration for morin was 50 mM whereas 10 mM for MST-312. Morin and MST-312 were either used alone or in combination throughout this study.

Matrigel (BD, Cambridge, MA, USA), 200 ml was spread as a thick layer on wells of a 24-well plate and allowed to polymerize at 37°C for 15 min. Cancer cells (2×10⁴) grown in monolayer were trypsinized to single cells and plated on top of the pre-coated Matrigel. Plates were incubated at 37°C to allow cells to fully settle down before media was replaced with appropriate culture media containing 5% Matrigel. Cells were grown for 15 days; fresh growth media with Matrigel was replenished every two days. Images of representative fields were taken.

Mouse fibroblasts (NIH-3T3) were used as a chemoattractant, and grown in a 24-well plate in 2 ml of the DMEM/F12 media. Boyden chambers were prepared with 25 μl of 1:6 diluted Matrigel and allowed to incubate for 2 h to solidify. Each chamber received a different treatment: untreated, morin only, MST-312 only and morin plus MST-312. After cell synchronization, invasion was allowed to occur for 40 h. The cells were then fixed with 0.5% glutaraldehyde and stained with 5% toluidine blue for cell counting. Three different 40× microscope fields were used to quantify the invasion statistics when counting cells.

Monolayer cultures of respective cell lines at 80–90% confluence were lysed using 100 μl of RIPA buffer (Thomas Scientific Inc. Swedesboro, NJ, USA). Tris-glycine (Bio-Rad, Irvine, CA, USA) gels were loaded with 50–100 μg of lysates. After electrophoresis, the gel was transferred to a nitrocellulose membrane for 2 h. The membrane was blocked for 1 h in 5% BSA or 5% skim milk at 4°C. The membrane was then washed 3 times with 1X TTBS and incubated overnight with the primary antibody at 4°C. Primary antibodies of STAT3, pSTAT3 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). After incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP), the protein bands were developed with the chemiluminescent reagents.

Cells were processed according to the manufacturer's protocol for the TeloTAGGG Telomerase PCR ELISA kit (Roche, Orange, CA, USA. catalog no. 11854666910). Briefly, cell pellets were thawed in lysis reagent, incubated on ice for 30 min, and centrifuged at 16,000 g for 20 min at 4°C. Telomerase activity was immediately measured in the resultant supernatant using the telomeric repeat amplification protocol in which telomerase, if present in the cell lysate, adds telomeric repeats to the 3′ end of a biotin-labeled synthetic P1-TS primer. Samples were amplified by polymerase chain reaction (PCR), with P1-TS and P2 primers creating an elongated telomere. The PCR product was denatured and hybridized to a digoxigenin-labeled probe that detects telomeric repeats in a subsequent enzyme-linked immunosorbent assay (ELISA). Samples were considered positive for telomerase activity if the ELISA resulted in a background-corrected absorbance of ≥0.2 U, resulting in binary positive/negative data. Telomerase assays were performed three times independently and P-values <0.05 were considered statistically significant.

Approximately 500,000 colorectal cancer cells were washed with 1X PBS, trypsinized, and then transferred to a 15-ml tube. Cell suspensions were centrifuged, re-suspended in 2 ml 1X PBS, and then divided into two tubes of 0.5 ml each. One tube was used as an unstained control and the other was stained with 5 μl CD44 antibody (FITC Green; BD Biotech, San Jose, CA, USA) or CD133 antibody (PE Red; Miltenyl Biotec, San Diego, CA, USA). The tubes were vortexed briefly and incubated at room temperature for 15 min in the dark. Each tube was then washed with 3.5 ml 1X PBS and then centrifuged for 6 min. The supernatant was removed by aspiration, and the cells were re-suspended in 3 ml 1X PBS and subjected to FACS profiling at the UCLA FACS Core Laboratory.

The Stress and Apoptosis Signal Antibody Array kit was purchased from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA; catalog no. 12856). Each CRC cell line had the following treatments in this order: untreated, morin only, MST-312 only, and morin plus MST-312. Whole protein lysates were prepared using the provided lysis buffer from the kit. One hundred milliliters of each lysate were placed onto the membrane window of the antibody slide. The treated slide was incubated overnight at 4°C on an orbital shaker. The slide was then washed with 100 ml 1X array wash buffer and incubated on an orbital shaker for 5 min at room temperature. This procedure was repeated three more times. 1X Detection Antibody Cocktail (75 μl) was added to each of the 16-wells and the plate was covered with the provided sealing tape. It was incubated for 1 h at room temperature on an orbital shaker. Next, three wash cycles were performed and the slide was incubated for 30 min with 75 μl 1X HRP-linked streptavidin. The slide was washed and treated with Lumi Glo and peroxide. The Bio-Rad Gel Documentation system was used to take detailed pictures of the array using the Quantity One software using the ChemiDoc XRS function. ImageJ software was used to analyze the antibody array. All the array images were scanned and saved as JPEG files. We utilized the ImageJ software to quantify the expression levels of proteins. The quantified protein expression levels were presented as histograms with statistic significance.

The cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method. Briefly, the 5-FU chemo-resistant cell lines, HT-29 and SW620, were seeded in a 96-well plate (1,000 cells per well) and exposed to different concentrations of 5-FU or 5-FU plus 5 μM morin, or 5-FU plus 3 μM MST-312 in triplicate for 24 h. Additionally, in another set of experiments, cells were co-treated with 5 μM morin and 3 μM MST-312 with different concentrations of 5-FU (0, 0.1, 1, 2, 3 and 4 μM) for 24 h to obtain the optimum dose for combination treatment. Cells were washed twice with PBS and subsequently, MTT solution (5 mg/ml) was added to each well and the plate was incubated for 4 h at 37°C. The 96-well plates were wrapped with aluminum foil and gently swirled for 15 min at room temperature. The absorbance of the cell suspension was measured at 570 nm. The data obtained were calculated and were represented as hundredth of survival relative to controls. This experiment was repeated 3 times independently, and statistical analysis was done to obtain the final values.

Student's t-tests were used to evaluate the significance of changes in all combination treatment assays compared to controls. Differences were considered statistically significant at P<0.05.

To confirm the molecular functions of morin and MST-312, we tested two colorectal cancer cell lines which contain the constitutively phosphorylated STAT3 (pSTAT3) and activated telomerase, HT-29 and SW620. Morin inhibits STAT3 phosphorylation in a dose-dependent and time-dependent manner (16). Initially, we treated HT-29 and SW620 cells with morin at the concentration 50 μM for 24 h. After the treatment, we ran a western blot analysis to examine STAT3 phosphorylation status. As shown in Fig. 1A, STAT3 phosphorylation was inhibited in both HT-29 and SW620 cell lines whereas total STAT3 expression levels remained the same (Fig. 1A). Our data suggest that morin specifically inhibited STAT3 phosphorylation step in colorectal cancer cell lines.

Next we wished to determine the telomerase activity in HT-29 and SW620 cell lines. MST-312 is a synthetic compound that functions as a reversible telomerase inhibitor (17). To monitor telomerase activity, TRAP-PCR-ELISA assay was performed as described in Materials and methods. HT-29 and SW620 were treated with morin alone at a concentration of 50 μM for 24 h, MST-312 alone at a concentration of 10 μM for 24 h and morin and MST-312 combination for 24 h and were applied to the telomere PCR-ELISA assays. As shown in Fig. 1B, MST-312 treatment inhibited telomerase activity, average absorbance was clearly decreased from 0.98 to 0.47 (OD, 490–750) whereas morin slightly reduced the absorbance from 0.95 to 0.90 in HT-29 (Fig. 1B). Morin and MST-312 combined decreased the absorbance to 0.35. Similarly, MST-312 decreased the absorbance from 0.99 to 0.41 (OD, 490–750) while morin reduced it from 0.99 to 0.93 in SW620 cell lines. When morin and MST-312 were combined, telomerase activity was decreased to 0.24. Our results confirm that MST-312 inhibited telomerase activity in human colorectal cancer cells and when combined with morin, the inhibitory effects were further enhanced.

To quantify the cancer stem cell population, we chose CD133 as a biomarker. FACS profiling revealed a clear reduction of the CD133⁺ population in the cancer cells treated with morin and MST-312. Untreated HT-29 showed that 69.9% of cells were CD133 (+) (Fig. 2A). However, when we treated HT-29 with morin (50 μM for 24 h), the CD133-positive subpopulation was reduced to 63% (Fig. 2B). Similarly, MST-312 treatment (10 μM for 24 h) reduced the CD133 (+) population to 64.5% (Fig. 2C). Moreover, the CD133 (+) population was decreased to 57.9% after combined treatment with morin and MST-312 in HT-29 cells (Fig. 2D). Representative histograms are presented with the statistical significance.

We also observed the same CD133 (+) pattern from the metastatic colorectal cancer cell line SW620. In the untreated control SW620, 81.7% of cells were CD133 (+) (Fig. 2E). After treatment with morin (50 μM, 24 h), the CD133 (+) population was reduced to 57.5% (Fig. 2F). With MST-312 treatment, CD133 (+) was decreased to 69.1% (Fig. 2G). Notably, the combined treatment of morin and MST-312 showed enhanced reduction of CD133 (+) to 55.5% (Fig. 2H). Our results demonstrate that combined treatments with morin and MST-312 indeed reduced the CD133 (+) subpopulations in both primary and metastatic human colorectal cancer cell lines. Histograms of the CD133 (+) populations are presented with statistical significance.

As combined treatment of morin and MST-312 reduced CD133 positivity, we next examined the cell-level invasiveness of the colorectal cancer cells. To this end, we employed two cell invasion assays, tumorsphere formation assay and boyden chamber assays. In tumorsphere culture condition, we created a three-dimensional microenvironment by adding 5% Matrigel to the 24-well plates. As shown in Fig. 3, both HT-29 and SW620 cells formed tumorspheres in 7 days. However, when we treated cells with morin and MST-312, the tumorsphere forming capacity was significantly reduced. Untreated control HT-29 formed ~32 tumorspheres per wells (Fig. 3A). However, when cells were treated with morin and MST-312, the formation was reduced to 15 and 21 tumorspheres, respectively. The combined treatment of morin and MST-312 abolished tumorsphere formation from HT-29. We observed a similar pattern in the SW620. Untreated control cells formed 45 tumor-spheres per wells (Fig. 3B). Morin treatment resulted in a reduction to 32 tumorspheres and MST-312 treatment a reduction to 21 tumorspheres per well. When we treated SW620 with morin and MST-312 together, tumorsphere formation capacity was abolished.

In agreement with the tumorsphere formation, cell invasion assays also revealed that combined treatment inhibited cell invasiveness. On average, the number of cells invading from the boyden chamber decreased from 55 to 2 when compared to untreated control HT-29 and morin/MST-312-treated cells (Fig. 4A). In SW620, the average invaded cell numbers decreased from 75 of untreated control to 12 of morin/MST-312-treated cells (Fig. 4B). Our results suggest that morin/MST-312 combined treatment can efficiently reduce the cancer stem cell subpopulations from human colorectal cancer cells.

The findings that morin and MST-312 treatment inhibited CSC phenotype suggest that it may directly trigger certain signaling pathways. Therefore, we wished to determine how morin and MST-312 treatments elicit cellular stress and apoptosis responses from colorectal cancer cells. To assess the net effects of the treatments, we decided to monitor the changes in key signaling components related to cellular stress and apoptosis. To this end, we utilized the stress and apoptosis signaling antibody array kit (Cell Signaling Technology). By using this array, we were able to simultaneously interrogate 19 signaling molecules that are involved in the regulation of stress response and apoptosis. Target-specific capture antibodies were spotted in duplicate onto nitrocellulose-coated glass slides. Cancer cell lysates were incubated on the slide followed by a biotinylated antibody. The expression differences from the antibody arrays were presented as histograms with statistical significance of the differential expression proteins in HT-29 and SW620. As shown in Fig. 5, morin treatment on HT-29 enhanced the levels of BAD and p53 phosphorylation (Fig. 5A). BAD is a member of the Bcl-2 family and a regulator of the programmed cell death pathway (22). Tumor suppressor p53 plays an important role in cellular responses to DNA damage (23). Thus, morin treatment activated BAD and p53 and concurrently induced apoptosis in HT-29 cells. MST-312 treatment inhibited p53 phosphorylation on the contrary, indicating that MST-312 inactivates p53, possibly independent of morin pathway. When morin and MST-312 were treated in combination, p53, Chk2 and TAK1 phosphorylation were inhibited in HT-29. Chk2 kinase acts downstream of ATM/ATR and plays an important role in DNA damage check point control (24). TAK1 is a kinase that can be activated by TFG-β, bone morphogenetic proteins and other cytokines (25). Morin treatment attenuated p53, Chk2 and TAK1 phosphorylation which are important for DNA damage control and cell survival. Increased apoptotic effects of morin and MST-312 treatments might be through the impaired DNA damage repair system and suppressed cell survival signaling.

Morin treatment on SW620 enhanced the phosphorylation of BAD, p53 and SAPK (Fig. 5B). SAPK kinase is activated through a dual phosphorylation of Thr202 and Tyr204 in response to pro-inflammatory cytokines and genotoxic stress (26). BAD and p53 activation with morin treatment is similar to HT-29. MST-312 treatment inhibited caspase-3 cleavage and downregulated IκBα expression level in SW620. Caspase-3 protease exerts a pro-apoptotic function through cleavage of multiple targets (27). Caspase-3 is activated at Asp175. IκBα is targeted to the proteasome via phosphorylation at Ser32 and Ser36 (28). Morin and MST-312 combined treatment activated BAD, p53 and SAPK phosphorylation whereas inhibited caspase-3 cleavage and IκBα phosphorylation in SW620. The enhanced apoptotic effects may be result from the inhibited cytokine signaling required for cancer cell survival. This distinct subset of gene inhibition in caspase-3 cleavage and IκBα is possibly due to the cell line specific differences between HT-29 and SW620. Taken together, our data revealed the existence of distinct expression patterns from the stress and apoptosis genes responding to morin and MST-312 treatments in the colorectal cancer cell lines.

Human colorectal cancer cell lines were sub-grouped based on their growth inhibition (GI₅₀) values against 5-FU treatments in a previous study (29). Three subgroups were chosen, consisting of 5-FU sensitive, intermediate and resistant colorectal cancer cell lines. Two 5-FU chemo-resistant cell lines, HT-29 and SW620, were used in our study to investigate the effects of morin/MST-312 and/or 5-FU on cell viability. The GI₅₀ values of HT-29 and SW620 were 14.90 and 17.97 μM, respectively. The cytotoxic effects of 5-FU or morin plus MST-312 on two colon cancer cell lines were determined using the MTT assay. The cancer cells were treated with different concentrations of 5-FU (0, 1, 5, 10, 20 and 50 μM) alone or combined with 5 μM morin and 3 μM MST-312. We observed that 5-FU blocked the proliferation of the cell lines HT-29 and SW620 in a dose-dependent manner with 5 μM morin and 3 μM MST-312 co-treatments (Fig. 6A). 5-FU efficacy was enhanced to the extent that the IC₅₀ level was reduced to 0.5 μM for HT-29 and 1 μM for SW620 (Fig. 6B). Both 5-FU chemo-resistant cell lines became equally sensitive to 5-FU with the co-treatment of 5 μM morin and 3 μM MST-312. Our data suggest that the morin/MST-312 combination treatment as an approach for the better treatment of human colon tumors with the potentially enhanced chemo-sensitivity to 5-FU.

Morin and MST-312 treatment inhibited the CSC phenotype in human colorectal cancer cells. Next we wished to determine whether this effect holds true in other human cancers. To test this, we chose the human triple-negative breast cancer cell line, MDA-MB-231. It also contains constitutively activated STAT3 phosphorylated by JAK2 kinase at the site of Tyr705 (30) and activated telomerase. Morin (10 μM for 24 h) and MST-312 (10 μM for 24 h) were used alone or in combination. Untreated control and treated cells were subsequently applied to FACS analysis for CD44 (+) profiling (Fig. 7A). CD44 is a well-established biomarker for breast CSC population. When treatment was used, the CD44 (+) subpopulation was reduced slightly from 96.5% (CD44⁺ of the untreated control) to 92% (Fig. 7B). Similarly, MST-312 treatment reduced the CD44 (+) population to 94.9% (Fig. 7C). The combined treatment with morin and MST-312 reduced the CD44 (+) to 85.9% (Fig. 7D). These data suggest that the synergism of morin and MST-312 may be conserved in multiple human cancers.

In agreement with FACS analyses, wound healing studies also showed synergistic effects of morin and MST-312 treatments. MDA-MB-231 cells were pre-treated with either morin/MST-312 alone or in combination for 24 h at the same concentration (morin; 50 μM and MST-312; 10 μM). Afterwards, the cells were seeded onto 24-well plates and strait scratches were performed. We observed wound healing up to 48 h. As shown in Fig. 8, untreated control cells rapidly healed the wounds (Fig. 8A). However, morin and MST-312 treatments clearly inhibited the wound healing (Fig. 8B and C). The inhibition effect was enhanced upon morin and MST-312 combination treatment (Fig. 8D). Together, our results indicate that morin and MST-312 combination treatment can downregulate breast cancer stem cell phenotype.