Cucurbitacin B inhibits cell proliferation and induces apoptosis in human osteosarcoma cells via modulation of the JAK2/STAT3 and MAPK pathways

Abstract

Osteosarcoma (OS) is the most commonly diagnosed tumor of the bones in children and young adults. Even with conventional therapies the 5-year survival rate is ~65% in patients with OS. Considering the side effects and aggressiveness of malignant bone tumors, research is focussing on multi-targeted strategies in treatment. Cucurbitacin B, a triterpenoid compound has been demonstrated to induce apoptosis in various cancer cell types. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling cascades and mitogen activated protein kinases (MAPK) signalling cascades are critical regulators of tumorigenesis. The present study assessed the influence of cucurbitacin B on the viability and expression of MAPKs and proteins of the JAK2/STAT3 cascades in human OS cells (U-2 OS). Cucurbitacin B (20-100 µM) significantly reduced cell viability (P<0.05) and induced apoptosis, as assessed by MTT and Annexin V/propidium iodide staining, along with inhibiting cell migration. Gelatin zymography revealed supressed activities of matrix metalloproteinase (MMP-)2 and 9. Furthermore, cucurbitacin B effectively upregulated the apoptotic pathway and caused the effective inhibition of MAPK signalling and JAK2/STAT3 cascades. Multifold suppression of vascular endothelial growth factor by cucurbitacin B was also observed, indicating inhibition of angiogenesis. Thus, by downregulating major pathways-MAPK and JAK2/STAT3 and MMPs, cucurbitacin B has potent anti-proliferative and anti-metastatic effects that require further investigation with regards to cancer treatment.

Keywords: Janus kinase/signal transducer and activator of transcription pathway; apoptosis; cucurbitacin; mitogen activated protein kinases; osteosarcoma.

Figure 1.

Cucurbitacin B reduces the cell viability of U-2 OS cells. *P<0.05 vs. control; ^#P<0.05 vs. 20 µM. Data are presented as mean ± standard deviation, n=3. OS, osteosarcoma.

Figure 2.

Cucurbitacin B induces apoptosis of human osteosarcoma U-2 OS cells. Cucurbitacin B was observed to significantly increase the apoptotic cell count and reduce cell viability in U-2 OS cells. *P<0.05 vs. control; ^#P<0.05 vs. 20 µM. Data are presented as mean ± standard deviation, n=3. OS, osteosarcoma.

Figure 3.

Influence of Cucurbitacin B on the viability of U-2 OS cells under Hoechst staining. Hoechst staining using a fluorescence microscope (Nikon Eclipse TiS coupled with NIS-Elements imaging software) revealed a significantly increased number of cells under apoptosis following cucurbitacin B treatment. *P<0.05 vs. control; ^#P<0.05 vs. 20 µM. Data are presented as mean ± standard deviation, n=3. OS, osteosarcoma.

Figure 4.

Influence of cucurbitacin B on cell migration determined by a wound healing assay. Cucurbitacin B exposure significantly reduced cell migration of the U-2 OS cells over the wound area in a dose-dependent manner. *P<0.05 vs. control; ^#P<0.05 vs. 25 µM. Data are presented as mean ± standard deviation, n=6. OS, osteosarcoma.

Figure 5.

Cucurbitacin B reduces activities of MMP-2, MMP-9 and VEGF in U-2 OS cells. This was determined by (A) gelatin zymography following exposure to cucurbitacin B, indicating marked downregulation in the expressions of MMP-2 and −9, (B) quantified results from the gelatin zymography indicating a significant downregulation of MMP activity. (C) Western blot analysis indicates a marked reduction in the expression of MMP-2, MMP-9 and VEGF. *P<0.05 vs. control; ^#P<0.05 vs. 25 µM. Data are presented as mean ± standard deviation, n=3. MMP, matrix metalloproteinases; VEGF, vascular endothelial growth factor; OS, osteosarcoma; L1, control; L2, 20 µM cucurbitacin B; L3, 40 µM cucurbitacin B; L4, 80 µM cucurbitacin B; L5, 100 µM cucurbitacin B.

Figure 6.

Cucurbitacin B modulates the expression of apoptosis pathway proteins. Cucurbitacin B caused effective upregulation of caspase-3, −8 and −9 in addition to an increase in the expression of Bax and Bad, while suppressing the expression of Bcl-2 and Bcl-xL, demonstrated in (A) western blot analysis and (B) quantification of the results. *P<0.05 vs. control; ^#P<0.05 vs. 100 µM. Data are presented as mean ± standard deviation, n=3. Bcl-2, B-cell lymphoma 2; Bcl-xL, Bcl-2 extra large; Bad, Bcl-2-associated death promoter; Bax, Bcl-2 associated X; L1, control; L2, 25 µM cucurbitacin B; L3, 50 µM cucurbitacin B; L4, 100 µM cucurbitacin B.

Figure 7.

Cucurbitacin B downregulates the expression of MAPK signalling cascades. Cucurbitacin B exposure markedly inhibited the activation of JNK, ERK1/2 and p38, thus inhibiting the signaling pathway, demonstrated by western blot analysis. MAPK, mitogen activated protein kinases; JNK, c-Jun N-terminal kinases; ERK, extracellular signal-regulated kinases; p-JNK, phosphorylated-JNK; p-ERK, phosphorylated ERK; L1, control; L2, 25 µM cucurbitacin B; L3, 50 µM cucurbitacin B; L4, 100 µM cucurbitacin B.

Figure 8.

Cucurbitacin B downregulates the expression of JAK2/STAT3 signalling cascades. (A) Western blot analysis indicates that cucurbitacin B induced marked downregulation of JAK2 and STAT3 expression, thereby inhibiting the signaling cascade and (B) quantified results indicate a significant reduction in expression of JAK2/STAT3 signaling cascades. *P<0.05 vs. control; ^#P<0.05 vs. 100 µM. Data are presented as mean ± standard deviation, n=3. JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; L1, control; L2, 25 µM cucurbitacin B; L3, 50 µM cucurbitacin B; L4, 100 µM cucurbitacin B.