Tris DBA decreased the viability and increased the subG1 population of HCC and MM cells.
We deciphered the action of Tris DBA on the viability of HCC and MM cells by MTT assay. For this, we used HCC (HCCLM3, Huh7, HepG2), MM (U266), and bortezomib-resistant/sensitive MM (RPMI-8226) cell lines. All the cells responded to Tris DBA, which resulted in a significant decrease of viability thus indicating the cytotoxic effect of Tris DBA on both types of cancer cells (Fig. 1B). Flow cytometric analysis revealed that the treatment with Tris DBA significantly increased the subG1 cell population up to 80% in HCC and MM cells (Fig. 1C). However, the subG1 cell population is relatively less (30%) in bortezomib-resistant RPMI-8226 cells.
Tris DBA induced apoptosis of HCC and MM cells.
We performed FITC-Annexin V-PI staining to verify the action of Tris DBA is inducing apoptosis and observed ta dose-dependent elevation in the percentage of apoptotic cells in all the tested cell lines (Fig. 2A). The percentage of the apoptotic population in bortezomib-resistant MM cells were relatively lower than the sensitive counterpart.
Tris DBA altered STAT3 phosphorylation in tumor cells.
Initially, we examined the effect of Tris DBA on activation of STAT3 in HCC and MM cells. Tris DBA significantly reduced the constitutively activated STAT3 in U266 and HCCLM3 cells (Fig. 1B). RPMI-8226 has a basal level of phospho-STAT3Y705 expression and treatment of these cells with IL-6 triggered the phosphorylation of STAT3Y705 in time- and dosage-dependent manner (Fig. 1C, upper panels). The treatment with Tris DBA resulted in suppression of IL-6 driven STAT3 activation without a change in the total STAT3 expression (Fig. 1C, lower panel).
Tris DBA decreased nuclear localization and ablated the binding of STAT3.
Next, we deciphered the action of Tris DBA on the cellular distribution of STAT3 in HCCLM3 cells. Tris DBA downregulated the nuclear pool of STAT3 in HCCLM3 cells compared with vehicle-treated samples as demonstrated by the results of immunocytochemistry (Fig. 3A). Further, we prepared the nuclear extract of DMSO/Tris DBA treated cells and analyzed whether Tris DBA also influences the DNA interaction ability of STAT3. We observed a significant reduction in the DNA interaction ability of STAT3 in different tumor cells (Fig. 3B). We also observed an inhibition of IL-6 triggered DNA binding of STAT3 in RPMI-8226 cells (Fig. 3B), thus indicating that Tris DBA may interfere with STAT3 activation and subsequent gene transcription.
Tris DBA inhibited the phosphorylation of upstream kinases of STAT3 signaling.
Next, we measured the potential of Tris DBA treatment on the phosphorylation of upstream kinases regulating STAT3 signaling such as JAK1, JAK2, and Src in HCC and MM cells. We observed a significant decline in the constitutive activation of JAK1, JAK2, Src in U266 cells, and JAK2, Src in HCCLM3 cells (Fig. 4A). We also investigated the effect of Tris DBA on IL-6-induced phosphorylation of JAK1 and JAK2 in RPMI-8226 cells. We observed a substantial reduction in the IL-6 induced activation of JAK1 and JAK2 (Fig. 4B), thereby indicating that Tris DBA suppresses the activity of upstream kinases to modulate STAT3 functions.
Tris DBA altered the expression of SHP2.
We further determined the action of pervanadate on Tris DBA induced STAT3 inhibition in MM cells. The exposure to pervanadate reversed the Tris DBA induced STAT3 inhibition thereby indicating the involvement of protein tyrosine phosphatases (PTPs) (Fig. 4C). The action of Tris DBA on the cellular levels of major PTPs such as SHP1, SHP2, and PTP1B was measured. Interestingly, Tris DBA elevated the protein levels of SHP2 in a dosage- and time-dependent fashion (Fig. 4D). We also observed that treatment with Tris DBA increased the mRNA expression of SHP2, thus indicating that this protein may be regulated at the transcriptional level (Fig. 4E). However, a substantial change was not noted in the expression of SHP1 and PTP1B proteins (Fig. 4F).
Depletion of SHP2 reverses the Tris DBA induced STAT3 inhibition.
We transfected U266 cells with siRNA direct towards SHP2 to study the specificity of Tris DBA towards the STAT3 signaling pathway. The treatment of Tris DBA alone decreased STAT3 phosphorylation with a corresponding increase in SHP2. The deletion of SHP2 using siRNA resulted in the restoration of STAT3 phosphorylation and Tris DBA did not affect the STAT3 activation in SHP2-depleted cells (Fig. 4G). The cells treated with scrambled siRNA served as control. These results substantiated the role of SHP2 in mediating the STAT3 inhibitory actions of Tris DBA.
Tris DBA altered the expression oncogenic proteins.
We determined the action of Tris DBA on the expression of STAT3 driven proteins in MM (Fig. 5A) and HCC cells (Fig. 5B). Consistent with inhibition of STAT3 phosphorylation, Tris DBA downregulated the expression of oncogenic factors such as cyclin D1, Bcl-2, Mcl1, survivin with an upregulation of proapoptotic factor such as Bak protein in the tested cell lines. Furthermore, Tris DBA also promoted the cleavage of caspase-3 and PARP thus, indicating that cells are undergoing apoptosis (Figs. 5A and 5B).
Tris DBA significantly modulated the migratory and invasive potential.
Next, we measured the outcome of Tris DBA on cellular motility using invasion and migration assay systems. Tris DBA significantly suppressed the invasion of U266, RPMI-8226 (sensitive and resistant), and HCCLM3 cells in a concentration-dependent fashion (Fig. 6A). In addition, significant anti-migratory effects were observed in Tris DBA treated HCCLM3 cells (Fig. 6B).
Tris DBA did not display toxicity in preclinical studies.
We next determined the sub-lethal doses of Tris DBA to perform in vivo tumor studies by measuring its toxicity. For this, male NCr nude mice were injected with Tris DBA (25, 50, 100, and 200 mg/kg/day) for eight days and experimental animals were regularly monitored. The mice-group which received DMSO (0.1%) served as control. We did not observe significant changes in Tris DBA treated group of animals relative to the DMSO treated group in terms of body weight, water and feed consumption (Fig. 7). Notably, no significant alterations were observed in the activity of liver function marker enzymes such as alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Fig. 7), thereby suggesting that Tri DBA did not exhibit significant toxicity in tested animals.
Tris DBA imparted antitumor effect in xenograft MM and orthotopic HCC mice models.
We investigated the anticancer activity of Tris DBA in xenograft MM and the orthotopic HCC mice models. We subcutaneously implanted U266 cells to the right flank of six weeks old athymic nu/nu female mice to establish the xenograft tumor model. When tumor size attained 0.25 cm, the mice were randomly divided into three groups (n = 6). The first group served as a control. The second and third groups received an intraperitoneal injection of Tris DBA (50 mg/kg, thrice a week; and 100 mg/kg, thrice a week, respectively). At the end of four weeks, we observed a significant reduction in the volume of tumors in the 100 mg/kg treated group (p < 0.01) (Fig. 8A).
To verify the antitumor effect of Tris DBA on solid tumors, we next established an orthotopic HCC mouse model as described in methods and randomly distributed into three groups. The first group was used as control. The second and third group animals were intraperitoneally injected with Tris DBA (50 mg/kg, thrice a week; and 200 mg/kg, thrice a week, respectively) for four weeks and the tumor burden was measured by a non-invasive technique of bioluminescence. Tris DBA significantly inhibited the tumor growth in both the groups (p = 0.0007) (Fig. 8B).
Tris DBA reduced the number of Ki67 + cells in tumor tissues from the xenograft MM mouse model.
We next processed the MM tumor tissue samples and performed immunohistochemical analysis to determine the proliferation index. Tumor tissues from Tris DBA treated mice displayed a marked reduction in the levels of Ki-67+ cells in a dose-dependent fashion (Fig. 9A). These results suggest that Tris DBA may also induce its antitumor activities by reducing the proliferation of cancer cells.
Tris DBA repressed the activation of STAT3 signaling proteins in tumor tissues.
We further analyzed the phosphorylation status of STAT3, JAK1, and JAK2 proteins in the tumor tissues of the xenograft MM mouse model. We observed a substantial reduction in the phosphorylation of STAT3, JAK1, and JAK2 in the tumor tissues of mice treated with 100 mg/kg of Tris DBA (Fig. 9B). These results establish that the lowering of tumor burden by Tris DBA could be due to the abrogation of the STAT3 signaling pathway.
Tris DBA modulated the expression of apoptosis-related proteins.
We profiled the apoptosis-related proteins (Bcl-2, Bcl-xL, Survivin, PARP, and procaspase 3) in tumor tissues of the xenograft MM mouse model. We observed a marked decrease in Bcl-2, Bcl-xL, Survivin, full-length PARP and procaspase-3 (Fig. 9C), thus indicating that Tris DBA can interfere with tumor development by inducing apoptosis in tumor cells.