br PIM downregulation exacerbates anti
3.4. PIM3 downregulation exacerbates anti-neoplastic activities of bufothionine
Based on the aforementioned results, experiments were designed to test the eﬀect of PIM3 downregulation in the anti-neoplastic activities against GC cells. At first, the expression of PIM3 was decreased by targeted siRNA treatment (Fig. 4A). Fig. 4B shows that bufothionine reduced cell viability to a larger proportion in both MKN28 and AGS 3X FLAG Peptide afterPIM3 knockdown. The results from LDH leakage assay also supported the finding that the cytotoxicity of bufothionine on MKN28 and AGS cells was more profound followingPIM3 knockdown (Fig. 4C). Flow cytometry results showed that PIM3 knockdown enhanced the apoptosis eﬀect of bufothionine in GC cells (Fig. 4D). Further, PIM3 knockdown exacerbated the bufothionine-induced expression of clea-vage of caspase-3 (Fig. 4E). At the same time, PIM3 enhanced bu-fothionine-induced upregulation of Bax and downregulation of Bcl-2 in GC cells (Fig. 4E). In the subsequent experiments, GC cells were transfected with PIM3 overexpressing vector to elevate PIM3 expression by > 150% compared with parental cells (Fig. 5A). The results from CCK-8 assay showed that ectopic PIM3 expression markedly compro-mised bufothionine-induced cell death in both MNK28 and AGS cells (Fig. 5B). The destructive eﬀect of bufothionine in both GC cells lines was also decreased by ectopic PIM3 expression (Fig. 5C). Ectopic PIM3 expression significantly dampened the apoptosis-inducing eﬀect of bufothionine on MKN28 and AGS cells, which was also validated by
Fig. 6. Bufothionine suppresses xenograft tumor growth. The treatment started when the tumor reached 100 mm3 and continued for 30 days before the mice were sacrificed. A. Bufothionine significantly suppressed tumor growth in vivo. B. The tumor was harvested at the end of the experiment and its weight was documented. C. The TUNEL assay was performed to quantitatively analyze cell apoptosis in the tumor tissue. D. The expression of PIM3, cleaved caspase-3, Bax and Bcl-2 in tumor tissues was visualized by IHC assay. E. The protein levels of PIM3, cleaved caspase-3, Bax and Bcl-2 in tumor tissues were quantitatively analyzed by western blotting. **P < 0.01.
western blotting assay on cleaved caspase-3, Bcl-2 and Bax (Fig. 5E). These results indicated that PIM3 downregulation exacerbated the anti-cancer activities of bufothionine against GC cells.
3.5. The anti-growth activities of bufothionine against GC in vivo correlates with downregulation of PIM3
The anti-cancer activities of bufothionine against GC were also evaluated in a xenograft mouse model. Treatment with bufothionine for 30 days remarkably suppressed the tumor growth and tumor weight in a dosage-dependent fashion (Fig. 6A and B). Moreover, bufothionine
treatment promoted apoptosis in vivo in a dosage-dependent manner (Fig. 6C). The results from IHC assay also showed that bufothionine treatment promoted caspase-3 cleavage expression, downregulatedBcl-2 and upregulated Bax, which correlated with PIM3 downregulation (Fig. 6D). The results from western blotting assay also supported the findings that, bufothionine treatment activated casapsae-3, upregulated Bax, and downregulated Bcl-2 and PIM3 in a dosage-dependent manner (Fig. 6E). The general toxicity of bufothionine was assessed by mon-itoring the changes of mice weight, levels of hepatic enzymes and urea nitrogen level. As shown in Supplementary Fig. 1A, bufothionine treatment at the indicated dosage did not significantly alter the body weight of xenograft mice model. Meanwhile, there was no significant change in hepatic and renal functions (Supplementary Fig. 1B and C). Moreover, no histological change was found in the liver, lung, kidney, heart and spleen tissues (Supplementary Fig. 1D). These results in-dicated that bufothionine was well-tolerated by murine models.
Cinobufacini (termed as Huachansu in Chinese), is an animal-de-rived drug which has been employed in the treatment of human ma-lignancies . It has been found that cinobufacin suppresses pro-liferation and promotes apoptosis of GC cells [22,23]. In addition, Cinobufacin has been tried as an adjuvant treatment in the conven-tional chemotherapy in patients with GC, which showed that Cinobu-facin could increase the response rate and disease the control rate of chemotherapy in advanced GC [24,25].Despite several reports have shown that bufadienolides was the main bioactive components of Ci-nobufacin. Little is known about which compound mainly contributes its suppressive role in GC. The results of the present study revealed that one of the bufadienolide compounds in Cinobufacin, bufothionine, promotes cell death and apoptosis of GC cells. In addition, it was found that PIM3 is the primary target responsible for the anti-cancer activities of bufothionine in GC. To sum up, bufothionine aﬀected GC progression through down-regulating PIM3 level.