Triphala churna (THL) is a combination of three fruits that has been used for many years in India for the treatment of various diseases. There are now reports which indicate that THL can inhibit growth of malignant tumors in animals. However, the mechanisms by which THL mediates its anti-tumor actions are still being explored. Because vascular endothelial growth factor-A (VEGF) induced angiogenesis plays a critical role in the pathogenesis of cancer, we therefore investigated whether tumor inhibitory effects of THL or its active constituents are through suppression of VEGF actions. We herein report that THL and chebulinic (CI) present in THL can significantly and specifically inhibit VEGF induced angiogenesis by suppressing VEGF receptor-2 (VEGFR-2) phosphorylation. These results are of clinical significance as these inexpensive and non-toxic natural products can be used for the prevention and treatment of diseases where VEGF induced angiogenesis has an important role
Results and Discussion
There are now studies which indicate the therapeutic efficacies of THL in tumor bearing animals –. However, there is still no report indicating the effects of THL on VEGF induced angiogenesis –. We at first determined whether single oral dose of 100 mg/kg of THL could inhibit VEGF (250 ng) mediated angiogenesis in vivo in a well established mouse matrigel plug assay model –. This dose of THL was particularly selected as this dose demonstrated the highest efficacy in human malignant tumor bearing mice . In addition, we also did not observe any significant changes in the complete blood count, hepatic enzymes, cholesterol, blood sugar, blood urea nitrogen (BUN) and serum creatinine level with this dose of THL in mice when compared to normal controls (data not shown). On day 8, THL untreated plugs containing VEGF appeared dark red, Masson’s trichrome staining (endothelial cells stain red and the matrigel stain blue) and CD31 immunostaining demonstrated higher levels of endothelial cells in these VEGF containing THL untreated plugs (Fig. 2 A–D). In contrast, on Day 8, plugs containing VEGF removed from animals treated with THL for 7 days were pale in color and the endothelial cells were also significantly less in numbers (Fig. 2A–D). Similar results were observed in control plugs without VEGF removed from animals untreated with THL (Fig. 2A–D). These data confirmed that oral administration of THL could significantly inhibit VEGF induced angiogenesis in vivo.
Furthermore in vitro studies have indicated the anti-VEGF actions of GA and EA, two constituents of THL , . Since the bioavailability of these two compounds following ingestion of either fruits containing these two acids or in pure forms is poor , , , ,  and because we had observed significant suppression of VEGF induced angiogenesis following oral administration of THL in our in vivo model (Fig. 2), we therefore examined the plasma level of another major constituent of THL, CI following oral feeding of mice with THL. The plasma concentration of CI reached to 1952.67 ng/ml (2.04 μM) at 20 min after gavaging the mice with a single dose of THL (100 mg/kg) containing 6.8 mg of CI as detected by LC-MS/MS.
Because VEGF mediates its angiogenic actions by stimulating proliferation, migration, tube formation and endothelial cell permeability –, therefore in order to investigate whether THL could specifically inhibit these functions of VEGF in endothelial cells, we initially determined the non-toxic concentration of THL to be used for our in vitro experiments in HUVEC by examining the cytotoxic effects of various concentrations of THL (20–80 μg/ml) that were previously reported to inhibit tumor cell growth in vitro , , . In addition, we also determined the effect of 2 μM of CI on the viability of HUVEC as this concentration of CI was detected in the plasma of mice after orally feeding them with the VEGF inhibitory dose of THL (100 mg/kg). Our results indicated 40 μg/ml of THL to be the highest non-toxic concentration of THL and 2 μM CI had no effect on cell viability (Fig. 3A, B). Accordingly, we selected 40 μg/ml of THL and 2 μM of CI for further in vitro experiments.
We next examined the effects of non-toxic concentration of THL (40 μg/ml) and CI (2 μM) on VEGF induced proliferation, migration, tube formation and permeability in HUVEC. Our results indicated significant inhibition of VEGF (20 ng/ml) induced proliferation (Fig. 3 C, D), migration (Fig. 4 A–D) and tube formation (Fig. 5A–D) by these cells after treatment with THL or CI. In addition, THL and CI also significantly inhibited VEGF induced permeability in HUVEC (Fig. 5E). It is to be noted here that THL (40 μg/ml) or CI (2 μM) alone had no effects on proliferation, wound healing, tube formation and permeability of the endothelial cells (data not shown).
Furthermore as these actions of VEGF is mediated mainly through its VEGFR-2 –, therefore to elucidate the molecular mechanisms by which THL or CI inhibited VEGF functions, we investigated the effects of THL (40 μg/ml) and CI (2 μM) on VEGF (20 ng/ml) induced VEGFR-2 phosphorylation in HUVEC. Our results demonstrated that THL or CI significantly inhibited VEGF induced phosphorylation of VEGFR-2 (Fig. 5F).
Since our previous in vitro data suggested that THL and CI could significantly inhibit the important steps of VEGF induced angiogenesis (Fig. 3, 4, 5), therefore, we determined the effects of THL (40 μg/ml) and CI (2 μM) on VEGF mediated angiogenesis in CAM assay , , . All observations were made on Day 4 after addition of these compounds. There was no evidence of angiogenesis or inflammation on addition of the vehicle (PBS) in which THL or CI were dissolved (Fig. 6A, E). However, striking angiogenesis was evident after exposure to 250 ng of VEGF (Fig. 6B, E). On the contrary, significant inhibition of VEGF induced angiogenesis was observed following exposures to 40 μg/ml of THL or 2 μM of CI (Fig. 6C, D, E). THL or CI alone did not induce any inflammation nor had any effects on blood vessel formation (data not shown).
Taken together our results for the first time demonstrated that THL or CI present in THL can significantly inhibit VEGF induced angiogenesis via suppression of VEGFR-2 actions. Moreover unlike the other constituents of THL such as GA and EA, the plasma level of CI reached considerably after oral intake of THL and this level of CI in turn could significantly and specifically inhibit the actions of VEGF in vitro. These results thus suggest that CI present in THL mediate the anti-VEGF effects of THL in vivo and is also a potent inhibitor VEGF functions. However, there may be other untested constituents of THL, which may also possess anti-VEGF activities.
Finally, VEGF mediated neovascularization plays an important pathogenic role in various diseases –. The presently available anti-VEGF drugs not only have serious toxicities, but are also very expensive –. This necessitates development of newer and effective non-toxic and inexpensive anti-VEGF agents. Our present study suggests that THL or CI may fulfill this promise in future
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