Functional Characterisation of Anticancer Activity in the Aqueous Extract of Helicteres angustifolia L. Roots

Abstract

Helicteres angustifolia L. is a shrub that forms a common ingredient of several cancer treatment recipes in traditional medicine system both in China and Laos. In order to investigate molecular mechanisms of its anticancer activity, we prepared aqueous extract of Helicteres angustifolia L. Roots (AQHAR) and performed several in vitro assays using human normal fibroblasts (TIG-3) and osteosarcoma (U2OS). We found that AQHAR caused growth arrest/apoptosis of U2OS cells in a dose-dependent manner. It showed no cytotoxicity to TIG-3 cells at doses up to 50 μg/ml. Biochemical, imaging and cell cycle analyses revealed that it induces ROS signaling and DNA damage response selectively in cancer cells. The latter showed upregulation of p53, p21 and downregulation of Cyclin B1 and phospho-Rb. Furthermore, AQHAR-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved PARP, caspases and Bax. Anti-apoptotic protein Bcl-2 showed decrease in AQHAR-treated U2OS cells. In vivo xenograft tumor assays in nude mice revealed dose-dependent suppression of tumor growth and lung metastasis with no toxicity to the animals suggesting that AQHAR could be a potent and safe natural drug for cancer treatment.

Fig 1. Selective cytotoxicity of AQHAR in human cancer cells.

(A) Cell viability of human cancer and normal cells treated with indicated doses of AQHAR. Human osteosarcoma (U2OS) cells showed dose-dependent decrease in viability; normal fibroblasts (TIG-3) were not affected by the equivalent doses (B) Phase contrast images of human cancer and normal cells showing toxicity of AQHAR (50 μg/ml) to cancer cells only. (C and D) Dose dependent reduction in colony forming efficacy of AQHAR-treated U2OS cells. Results are represented as mean ± SD of three independent experiments. ***p<0.001 denotes statistically significance difference between the control and treated groups. AQHAR, aqueous extract of Helicteres angustifolia L. root.

Fig 2. Cell cycle analysis of control and AQHAR treated cancer and normal human cells.

Cell cycle distribution of control and AQHAR-treated U2OS (A and C) and TIG-3 (B and D) cells are shown. Results from three independent experiments are represented as mean ± SD. *p<0.05 denotes statistically significance difference between the control and treated groups.

Fig 3. Induction of apoptosis by AQHAR in U2OS cells.

Control and AQHAR-treated cell populations were examined for Annexin-V expression by cytometric analysis. As shown, increase in apoptotic cells was observed in cultures treated with increasing doses of AQHAR.

Fig 4. Effect of AQHAR on key regulators of cell cycle arrest and apoptosis in U2OS and TIG-3 cells.

Cells were treated with AQHAR for 24–48 h and subjected to (A) Western blotting and (B) immunostaining for cell cycle regulatory proteins (p53, p21, Cyclin B1 and p-Rb). (C) Western blot analysis revealed activation of apoptotic signaling in U2OS cells. Quantitation of the results from three independent experiments is shown as mean ± SD with statistical significance as *p<0.05 between the control and AQHAR-treated cells.

Fig 5. Induction of DNA damage and ROS by AQHAR treatment.

AQHAR-treated cells exhibited increase in the γH2AX foci, signifying induction of DNA damage (A) and ROS (B) in U2OS cells. (C) Neutral comet assay in control and AQHAR-treated U2OS and TIG-3 cells. Mean percent Tail DNA and Tail moment was calculated using CASP Software. *p<0.05 denotes statistically significant difference between the control and treated U2OS cells. H₂O₂ (100 μM) was used as a positive control.

Fig 6. Cytotoxicity of curcubitacin B on cancer and normal human cells.

(A) Schematic representation showing the effect of AQHAR on cancer cells by induction of oxidative stress (ROS) and DNA-damage leading to activation of growth arrest and apoptosis signaling. (B) Effect of curcubitacin B on a variety of human cancer and normal cells, ***p<0.001 denotes statistically significant difference between the control and treated groups. (C) Phase contrast images of control and curcubitacin B-treated human osteosarcoma (U2OS) and normal (TIG-3) cells showing the toxicity to both. (D) Cell cycle analysis showing the arrest of U2OS and TIG-3 cells in G2/M phase in response to curcubitacin B treatment. (E) Cucurbitacin B-treated U2OS cells show decrease in phosphorylated RB (pRB) and increase in p53 at doses as low as 0.025 μM. Similar effects were observed by immunostaining in both cancer (U2OS) and normal (TIG-3) cells (F).

Fig 7. Tumor-suppression by AQHAR treatment in nude mice tumor xenograft assays.

(A) Body weight changes and tumor volume in control and AQHAR-fed mice. (B) Images of dissected tumors and their average weight in control and AQHAR-treated groups. (C) Anti-angiogenic effect of AQHAR in HT1080 xenograft tumors as determined by immunohistochemical staining for CD 31, and hematoxylin and eosin. (D) Effect of AQHAR on lung metastasis showing decreased in number of lung tumors in AQHAR-treated group. (E) Change in body weight during tail vein lung metastasis experiments. *p<0.05, **p<0.01 and ***p<0.001 denote the statistically significance between the control and treated groups.

Fig 8. Effect of AQHAR on hnRNP-K and mortalin, tumor metastasis mediating proteins, in A549 and U2OS cells.

(A) Western blot analysis for hnRNP-K and mortalin showing decrease in hnRNP-K. (B) Immunostaining of showing decrease in nuclear hnRNP-K and clustering of mortalin staining. Results were represented as mean ± SD of three independent experiments. *p<0.05 denotes the statistically significant difference between the treated and control groups.