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a b
Figure 5: Effect of WS treatment on inhibition of lung metastasis in nude
mice. HE staining of lung tissue sections after treatment with or without
WS for 8 weeks (×20). (a) WS treated mouse lungs showed no tumor
metastasis (n = 10); (b) six of ten mice showed tumor metastasis to the lungs,
with a total of 12 metastatic foci in the blood vessels and the parenchyma of
the lungs in control mice. WS: Withania somnifera
Figure 4: Effect of WS on suppression of growth of xenografted
MDA-MB-231 cells in nude mice. The bars represent the means ± standard
deviations of tumor size (mm ) (n = 10). The highest reduction (60%)
3
relative to the untreated control was shown after 8 weeks of WS
treatment (P < 0.05). Student’s t-test was used to assess significant
differences between treated groups and the untreated control group.
WS: Withania somnifera

Figure 7: Effect of WS treatment on the regulation of cytokine expression.
Quantitative reverse transcription-polymerase chain reaction was used to
measure cytokine expression in cells treated or not treated with 50 μg/mL
WS. WS: Withania somnifera

the optimal dose. In addition, the in vitro cytotoxic
concentration, ranging between 50 and 100 μg/mL,
gave us an idea about the dose. In a previous study, WS
root extract inhibited lung metastasis of xenografted
MDA-MB-231 cells at a dose of 8 mg/kg body weight,
[19]
administered 3 times a week for 4 weeks. This dose
is 37.5 times less than the dose used in our current
study. There is no obvious explanation for the difference
in the two doses. Differences in the source of roots,
age of roots, and extraction yield may contribute to
Figure 6: Effect of WS on inhibition of cytokine/chemokine expression.
WS: Withania somnifera different dose-responses when using crude plant extracts.
However, the WS extract, at a dose of 150 mg/kg/day
Our current data are consistent with those reported by for 155 days, caused a 23% reduction in development
others. A root extract of WS showed dose-dependent of mammary tumors in rats administered the carcinogen,
[17]
inhibition of tumor growth and metastatic lung nodule methylnitrosourea. [23]
formation with the minimal toxicity to mice. The In transgenic (MMTV/Neu) mice that received a diet
[17]
extract apparently inhibited cancer metastasis through containing the extract (750 mg/kg of diet) for 10 months,
inhibition of the epithelial-mesenchymal transition mice in the treated group (n = 35) had an average of 1.66
(EMT). Furthermore, withaferin A treatment of mammary tumors, and mice in the control group (n = 33)
MCF-10A cells inhibited EMT and in mice, reduced had 2.48, a reduction of 33%. Moreover, in treated
mammary cancer growth, effects of which were mice, WS caused a 50% reduction in the expression of
associated with reduced vimentin expression. In CCL2. [24]
[22]
the present study, the oral dose of WS extract used to
inhibit tumor metastasis to the lungs was 300 mg/kg/day WS caused in vitro and in vivo inhibition of breast
body weight. This dose was extrapolated from the cell cancer MDA-MB-231 cells and caused a signifi cant
culture experiments regarding the effect of WS extract reduction in expression of the cytokine, CCL2, a marker
on MDA-MB-230 cells. This dose was selected based of the metastasis of breast cancer to other organs. These
on a pilot study involving a range of doses to estimate results warrant further studies to assess the underlying

98 Journal of Cancer Metastasis and Treatment ¦ Volume 1 ¦ Issue 2 ¦ July 15, 2015 ¦