Antitumor Activity of Water Extracts From Cordyceps Militaris in NCI-H460 Cell Xenografted Nude Mice

1. Introduction 

Cordyceps is a stroma of the fungus that exists as a composite parasite on the larva of some insects and the dead caterpillars. It is sweet in taste and neutral in nature, acting on the Lung and Kidney meridians, and has the effects of reinforcing the lung and the kidney, arresting bleeding, and dispelling phlegm. Cordyceps has been used in chronic cough and asthma, hemoptysis in phthisis, impotence and emissions, aching of loins and knees [1], and contains five main nutrients and trace elements such as cordycepin, cordycepic acid, ophiocordin, glucan and complex polysaccharides. Studies investigating active ingredients are still proceeding [2].

Cordyceps militaris, a species of Cordyceps, is native to Korea. C. militaris contains large amounts of cordycepin and is known to have antibacterial, antitumor, immune-enhancing and addiction-detoxification effects [3]. Global attention on C. militaris has been increasing given its antitumor and immune enhancing activity. Ergosterol peroxide extracted from C. militaris has shown a strong activity against a Korean gastric cancer cell line in vitro [4]. Furthermore, water soluble fraction of C. militaris showed cytotoxic activity on three kinds of human cancer cell lines: stomachic adenocarcinoma, colorectal adenocarcinoma, and hepatocellular carcinoma. The presumed active component in the extract was cordycepin [5]. One study, however, has suggested that many ingredients in C. militaris have a stronger antitumor effect compared with cordycepin [2]. Recently, an in vitro assay to determine the antitumor mechanism of C. militaris reported that apoptotic events in A549 cells due to water extract of C. militaris were mediated via diminished telomerase activity through the inhibition of human telomerase reverse transcriptase transcriptional activity [6].

Base on these results, we performed an in vivo trial to evaluate the antitumor effect of administration of water extracts from C. militaris to lung cancer xenografted mice. We measured the volume of xenografted tumor, weight of each organ and survival time of mice. Cells were also inspected to determine if there was any kind of pathologic change.

2. Materials and Methods 

2.5. Changes in tumor volume 

The sizes of tumor mass were recorded twice a week following measurement with a digital caliper. The tumor volume was estimated according to the formula:

Vmean is the mean tumor volume in mm3, A is the largest tumor diameter and B is the smallest tumor diameter in mm.

Based on the regression of tumor volume, the antitumor activities of treatment were expressed by inhibition rate:

Vcontrol and Vtreatment are tumor volume of control group and treatment respectively. The tumor weights were also measured on the final day of the experiment after sacrifice of animals and excision of the tumor mass.

2.6. Mean survival time and percentage increase in lifespan 

To compare the lifespan of mice xenografted with NCI-H460 tumor fragments, mean survival time was estimated from the day when the tumor volume reached 1000 mm3 as described previously, and percentage increase in lifespan (%ILS) was calculated according to the equation:

Dcontrol and Dtreatment are the mean survival day of mice in control and treatment group, respectively [9, 10].

3. Results 

3.1. Changes in body weight 

Changes in body weight of each group are shown in Figure 1. The mean body weights were 23.67-25.89 g in the 150 mg/kg/day WECM-treated group and 24.24-25.99 g in the 300 mg/kg/day WECM-treated group. But there were no significant differences when compared with the control group (24.10-26.43 g).

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Figure 1. Changes in body weights of nude mice bearing NCI-H460 tumors under treatment with water extracts of Cordyceps militaris (WECM).

3.2. Changes in tumor volume 

As shown in Figure 2, WECM treatment (150 and 300 mg/kg/day) resulted in slight growth inhibition of NCI-H460 cell-transplanted solid tumor compared with the control group, but there were no significant difference. On day 22, the mean tumor volume of the 300 mg/kg/day WECM-treated group (1771.5 mm3) was lower than that of control group (2150.81 mm3), but there were no significant intergroup difference.

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Figure 2. Changes in tumor volume of nude mice treated with water extracts of Cordyceps militaris (WECM).

3.3. Inhibition rate (IR) on tumor volume 

The IR of each group is shown in Table 1. In the whole experiment, the IR of the 150 mg/kg/day WECM-treated group was 94.73-75.08% and that of the 300 mg/kg/day WECM-treated group was 85.81-73.81%.

Table 1. Inhibition rate on tumor volume in NCI-H460 tumor-bearing mice*

	Treatment	1 d	5 d	8 d	12 d	15 d	19 d	22 d
	Saline	100	100	100	100	100	100	100
	WECM
	150 mg/kg/day	95.13	94.73	75.08	80.23	86.06	90.55	89.10
	300 mg/kg/day	94.03	88.00	73.81	80.65	85.81	84.50	82.36

* Data presented as %. WECM = water extracts of Cordyceps militaris.

3.4. Final tumor weights and volume 

Tumor weights and volumes of each group on day 22 are shown in Table 2. Tumor weight and volume of the control group was 3.046 ± 0.793 g and 3.379 ± 1.184 cm3, respectively. Tumor weight and volume of 150 mg/kg/day WECM-treated group were 2.435 ± 0.586 g and 2.288 ± 0.510 cm3, respectively. Tumor weight and volume of 300 mg/kg/day WECM-treated group were 2.175 ± 0.475 g and 2.069 ± 0.440 cm3, respectively. Compared with the control group, both tumor weight and volume of in two WECM-treated groups were decreased. The 300 mg/kg/day WECM-treated group showed significant decrease compared with control group (p < 0.05; Table 2).

Table 2. Tumor weights in mice xenografted with NCI-H460 cells on day 22*

	Treatment	Tumor weight (g)	Tumor volume (cm3)
	Saline	3.046 ± 0.793	3.379 ± 1.184
	WECM
	150 mg/kg/day	2.435 ± 0.580	2.288 ± 0.510
	300 mg/kg/day	2.175 ± 0.475†	2.069 ± 0.440†

* Data presented as mean ± standard deviation † Statistical significant defined as p < 0.05 as compared to control saline group. WECM = water extracts of Cordyceps militaris.

3.5. Mean survival time and %ILS 

Mean survival time and %ILS are shown in Figure 3 and Table 3. The mean survive time of control group was 18.43 ± 2.23 days. The mean survival time of the 150 mg/kg/day WECM-treated group was extended to 19.43 ± 2.44 days, increasing %ILS by 5.42%. The mean survival time of 300 mg/kg/day WECM-treated group was 21.86 ± 3.53 days, increasing %ILS by 18.61%. However, the difference were not statistically significant.

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Figure 3. Mean survival time in NCI-H460 tumor-bearing mice treated with water extracts of Cordyceps militaris (WECM)

Table 3. Percentage increase in lifespan (% ILS) of NCI-H460 tumor-bearing mice

	Treatment	Mean survival time (d)	% ILS
	Saline	18.43 ± 2.23	0
	WECM
	150 mg/kg/day	19.43 ± 2.44	5.42
	300 mg/kg/day	21.86 ± 3.53	18.61

WECM = water extracts of Cordyceps militaris.

3.6. Histopathological findings and organ weights 

Absolute organ weights of the kidney, liver, spleen, heart and lung are shown Table 4. Both the 150 and 300 mg/kg/day WECM-treated groups showed a marked decrease in liver weights (p < 0.05). However, light microscopic histopathological examination showed no specific lesion in liver tissue of mice in neither WECM-treated groups nor control group (Figure 4).

Table 4. Organ weights of NCI-H460 tumor-bearing mice on the final day

			Absolute organ weights (g)
	Treatment	Body weight	Epididymis		Kidney		Testis		Liver	Spleen	Heart	Lung
			L	R	L	R	L	R
	Saline	26.429 ± 1.149	0.036 ± 0.006	0.034 ± 0.005	0.270 ± 0.034	0.269 ± 0.036	0.091 ± 0.006	0.091 ± 0.005	1.894 ± 0.173	0.454 ± 0.157	0.156 ± 0.015	0.203 ± 0.035
	WECM	26.177 ± 1.535	0.035 ± 0.011	0.033 ± 0.005	0.246 ± 0.029	0.253 ± 0.027	0.091 ± 0.014	0.089 ± 0.009	1.867 ± 0.247	0.409 ± 0.117	0.160 ± 0.009	0.193 ± 0.019
	150 mg/kg/day
	WECM	26.049 ± 1.010	0.043 ± 0.016	0.043 ± 0.016	0.258 ± 0.035	0.250 ± 0.031	0.096 ± 0.009	0.099 ± 0.007	1.716 ± 0.112	0.397 ± 0.048	0.171 ± 0.013	0.223 ± 0.033
	300 mg/kg/day

			Relative organ weights (%)
	Treatment	Body weight	Epididymis		Kidney		Testis		Liver	Spleen	Heart	Lung
			L	R	L	R	L	R
	Saline	26.429 ± 1.172	0.131 ± 0.022	0.120 ± 0.020	0.991 ± 0.105	1.027 ± 0.123	0.326 ± 0.029	0.327 ± 0.033	7.140 ± 0.455	1.655 ± 0.688	0.560 ± 0.051	0.762 ± 0.116
	WECM	25.823 ± 1.533	0.135 ± 0.043	0.134 ± 0.026	0.904 ± 0.057	0.944 ± 0.081	0.331 ± 0.045	0.330 ± 0.031	6.926 ± 0.320	1.544 ± 0.418	0.612 ± 0.038	0.759 ± 0.101
	150 mg/kg/day
	WECM	26.039 ± 0.945	0.163 ± 0.058	0.175 ± 0.065	0.996 ± 0.123	0.969 ± 0.111	0.365 ± 0.030	0.379 ± 0.022	6.615 ± 0.315*	1.532 ± 0.160	0.661 ± 0.053	0.852 ± 0.122
	300 mg/kg/day

* p <0.05. WECM = water extracts of Cordyceps militaris.

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Figure 4. Histopathological examination of liver tissue of NCI-H460 cell-xenografted mice treated with (A) saline and (B) 300 mg/kg/day water extracts of Cordyceps militaris (H&E; original magnification, 160×).

4. Discussion 

C. militaris is well known to contain cordycepin and ergosterol peroxide, two ingredients with antitumor effects [4]. Cordycepin was reported to be 3'-deoxyadenosine, a derivative of adenine, which could have antitumor effects by replacing adenine in DNA and RNA [10]. Ergosterol peroxide is reported to exhibit cytotoxic activities against tumor cells [11].

However, clinicians in the East have practiced using whole C. militaris, the evaluation of the anti-tumor effect of whole C. militaris would be more practical. To this end, an assay showing that water extract of C. militaris induced apoptosis and inhibited telomerase activity in human lung carcinoma cells has been published [6]. This activity occurred by activating the intrinsic caspase pathway along with the death receptor-mediated extrinsic pathway and was associated with a down regulation of telomerase activity due to a decrease of human telomerase reverse transcriptase expression [6].

Human tumor xenografts in immunodeficient animal models provided a means to evaluate potential antitumor drugs in preclinical studies and are applicable for studying many different types of human malignancies [12]. Thus we also conducted an in vivo experiment on nude mice with NCI-H460 cell-xenografted tumor for 28 days. These tumors were treated with WECM (150 and 300 mg/kg/day). Time-dependent changes of tumor volume were measured and excised tumor volume was measured on the final day. Results showed that the tumors were suppressed by WECM treatment but did not shrink in a dose-dependent manner.

WECM inhibited the NCI-H460 tumor growth in nude mice throughout the experimental period. WECM treatment (150 and 300 mg/kg/day) prolonged the mean survival time and increased %ILS compared with the control group. The survival time of the 150 mg/kg/day WECM treatment group was extended to an average of 19.43 ± 2.44 days and increased %ILS by 5.42%. Also, the mean survival time of the 300 mg/kg/day WECM treatment group was 21.86 ± 3.53 days, an 18.61% increase in %ILS. Intraperitoneal injection of C. militaris extracts into ICR mice bearing at least 180 sarcoma cells was shown to prolong the lifespan of mice from 52.4 to 132% [2]. Findings from this study and previous results are similar in that the lifespan of mice was increased even though the methods were different. However, the difference in lifespan extension between two studies may indicate that different administration methods could cause differences in efficacy.

The results showed that WECM inhibited the growth of NCI-H460 cell-transplanted solid tumor. Compared with the control group, the treatment group showed tumor growth inhibition from day 8 to day 22. However, this trend was not significant statistically. On day 22, the volume of the solid tumor was 2.069 ± 0.440 cm3 in the 300 mg/kg/day WECM-treated group and 3.3379 ± 1.184 cm3 in the control group. Tumor weights were 2.175 ± 0.475 g in the 300 mg/kg/day WECM-treated group), 2.435 ± 0.58 g in the 150 mg/kg/day WECM-treated group and 3.046 ± 0.793 g in the control group (Table 2). Previously, an in vitro study demonstrated that an ethanol extract of C. militaris produced an inhibitory effect of up to 65.5% in A549 human lung cancer cells [13], indicating that C. militaris may inhibit cancer growth. Numerical differences between this pervious study and the present data may be due to the differences in methodology (in vitro vs. in vivo) and extraction technique (ethanolvs. water). The underlying reasons for these differences should be elucidated to allow further exploitation of the use of C. militaris in clinical practice.

There were no differences in body weight, gross toxicity and organ weight between WECM-treated group and control group. There was no significant change in absolute organ weights, except in the liver. The results of in vivo studies implied that WECM may have potential as a growth inhibitor of solid tumor induced NCI-H460 without marked side effects. These results suggest that WECM may have the possibility to be a useful anticancer agent for therapy in human lung cancer.

Most of the results of this study indicate that WECM shows an antitumor effect in animal models but some are not significant. Although no significant pathologic changes in liver tissue were observed, the reasons for increasing relative liver weight needs to be further investigated.

In summary, this study showed that WECM may suppress the growth of a human lung cancer cell mass and increase the lifespan of nude mice. Results of this trial may stimulate more studies into WECM to determine its clinical use in cancer patients.