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The antitumor activities of Cucurbitacin Liposome for 

Injection bothin vitro

andin vivo

Junwei Wang, Xiaomian Zhou, Yinglin Cao, Jinfang Xiao, Enlong Ma*, Yihui Deng, Dawei Chen

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China

In this study, the antitumor activities of Cucurbitacin Liposome for Injection (CLI) were investigated in vitro and in vivo.   In vitro,

the antitumor effects of CLI on the growth of cultured human cervical carcinoma HeLa cells, human hepatoma cells BEL-7402 and

HepG2, and murine sarcoma S180 cells were determined by MTT assay and trypan blue dye-exclusion assay.   In vivo, antitumor

activity of CLI was investigated using H22

hepatocellular carcinoma cells, murine sarcoma cell S180 and Lewis lung carcinoma cells.

Flow cytometry was used to investigate the distribution of cells in each phase of cell cycle in S180 cells. CLI, at a low concentrations

(1-100 nM), had a significant inhibitory effect on proliferation of HeLa, BEL-7402, HepG2 and S180 cells  in a dose-dependentmanner. CLI at 0.0275, 0.055, 0.11 mg/kg inhibited tumor growth in mice of S180, H

22and Lewis lung carcinoma cells, respectively.

Cell cycle analysis demonstrated G2/M phase arrest in S180 cells following 24 h exposure to CLI. In conclusion, CLI has signifcant

antitumor effects in vivo and in vitro probably due to induction of G2/M cell cycle arrest.

 Key words: Cucurbitacin Liposome for Injection (CLI), antitumor, MTT assay, G2/M arrest

* Author to whom correspondence should be addressed. Tel:

+86-24-23986302; E-mail: maenlong@hotmail.com

Received: 2006-12-06 Accepted: 2007-05-11

In the search for new cancer therapeutics,

herbs being used in traditional medicines for cancer 

treatment are promising. Cucurbitacins are a class

of compounds charactered as highly oxygenated

tetracyclic triterpenes, which are predominantly found

in the cucurbitaceae family, but are also present in

several other families of the plant kingdom [1].

Many pharmacological and clinical investigations

have verified that cucurbitacin B (CuB) and

cucurbitacin E (CuE) possess various pharmacological

activities, such as, antitumor, anti-hepatitis and

immunopotentiating effects [2, 3]. The cucurbitacin

 preparation used clinically contains mostly CuB and

CuE, which are obtained from the calyx melo of  

Cucumis melo L ., a Chinese medicinal plant [4] that is

effective against chronic hepatitis and primary liver 

carcinoma [3].

In view of the data related to the antitumor 

activities of cucurbitacins, a new cucurbitacin

 preparation, Cucurbitacin Liposome for Injection

(CLI), was developed, and the antitumor activities of 

CLI were observed in the present study.

Material and methods

 Preparation

Cucurbitacin Liposome for Injection (CLI)

was supplied by Pharmaceutics Teaching and

Research Division, School of Pharmacy, Shenyang

Pharmaceutical University (Shenyang, China), which

Fig. 1 Molecular Structure of cucurbitacin B (C32

H46

O8. MW:

558.70)

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contains mostly CuB (Fig. 1) (85 %). CLI was diluted

with 5 % Glucose Injection (GS) (Shenyang Zhiying

Pharmacutical Factory, Shenyang, China) in in vivoexperiments, and with RPMI-1640 medium (Sigma,

St. Louis, MO, USA) supplemented with 10  % heat-

inactivated fetal bovine serum (FBS) (Hyclone, Logan,

Utah, USA) in in vitro experiments.

Tumor cell lines

HeLa human cervical carcinoma cells were

obtained from the American Type Culture Collection

(ATCC, #CRL, 1872, Rochville, MD, USA); human

hepatoma cells BEL-7402 and HepG2 cells were  purchased from the Chinese Academy of Medical

Sciences (Beijing); S180 murine sarcoma cell were

 purchased from the China Medical University Tumor 

Research Institute (Shenyang). Cells were cultured

in RPMI-1640 medium supplemented with 10  %

heat-inactivated FBS, 2 mmol/L glutamin (GIBCO,

Gaithersburg, Maryland, USA), 100 KU/L penicillin

and 100 g/L streptomycin (GIBCO, Gaithersburg,

Maryland, USA) at 37  ºC in 5 % CO2.

H22 hepatocellular carcinoma cells and Lewis lungcarcinoma were purchased from the China Medical

University Tumor Research Institute (Shenyang) and

the Academy of Military Medical Sciences of Beijing,

respectively. Recovery and passage were performed in

our laboratory.

 Animals

Male KM mice were obtained from Shenyang

Pharmaceutical University Animal Research Center 

(Shenyang, China). Male C57BL/6 mice were purchased from The Academy of Military Medical

Sciences of Beijing. The mice were housed in a

temperature- and light-controlled environment and

were allowed free access to food and water.

Cell growth inhibition assay

HeLa, BEL-7402, and HepG2 tumor cells were

 plated at 5 × 103 cells per well in 96-well culture

 plates and incubated for 24 h at 37 °C. After the cells

adhered, CLI (3-100 nM) or Cisplatin (Jinzhou Jiutai

 pharmaceutical Co.Ltd.,Jinzhou ) (50 μM) was then

added to growth medium. As a control group, the

vehicle, DMSO (end-concentration of 0.1  %), wasadded to growth medium. The medium was exchanged

daily. At 48 h and 72 h, cell growth was determined by

MTT assay. The percentage of cell growth inhibition

was calculated as follows:

Inhibition (%) = [ A492

(control)–  A492

(drug)]/ A492

(control) ×100

S180 cells were plated at 2 × 104 cells per well in

24-well culture plates, and then CLI (1-30 nM) was

added to growth medium. After 72 h, trypan blue was

added into the cell suspension to differentiate deadcells from living cells. The number of living cells was

determined with the aid of a hemocytometer, and the

relative cell number was expressed as the ratio of the

living cell numbers in CLI-treatment group to that of 

control group.

Flowcytometric analysis

S180

cells (1 × 106) were seeded in cell culture

flasks and treated with CLI for 24 h at 37 °C. Cells

were washed with PBS, and then xed in 70  % ethanolat -20 °C. Fixed cells were suspended in 1 mL PBS,

and treated with RNase (100 μg/mL) at 37  °C for 30

min. The cell suspension was stained with propidium

iodide (50 μg/mL) at 4 °C for 15 min, and analyzed

using a ow cytometer (BD Biosciences, USA).

 Antitumor assay in vivo

The experiment was carried out according to

the method described by Xu and his coworkers [5].

H22 (2×

106

cells), S180 (2×

106

cells) or Lewis lungcarcinoma (2×106 cells) cells in 0.2 mL sterilized

saline were injected subcutaneously into the oxter 

of 7-week-old KM mice. 72 h after implantation,

animals were treated intravenously with  CLI

(0.0275, 0.055, 0.11 mg/kg) once a day for 10 days

or 5-fluorouracil (20 mg/kg) every other day for 10

days. The control group was treated with 5 % GS or 

with empty liposomes. Then the mice were sacriced

under ether anesthesia, and the tumor tissues were

isolated and weighed to determine antitumor effects

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of CLI. The percentage of tumor growth inhibition

was calculated as follows:

Inhibition (%) =[tumor weight (control)–tumor weight (drug)] / tumor weight (control)×100

 Statistical analysis

The differences between multiple groups were

assessed by one-way analysis of variance (ANOVA)

followed by the Scheffé’s multiple range test. Values

of  P less than 0.05 were considered to be signicant.

The data obtained were expressed as mean ± SD.

Results and discussion

Cell growth inhibition assay

After 48 h and 72 h treatment, CLI (3-100 nM)

inhibited the growth of HeLa, BEL-7402 and HepG2

tumor cells in a dose-dependent manner  (Fig. 2).Even at a lower concentration (1-30 nM), CLI had

signicant inhibitory effects on S180 cells.

Flowcytometric analysis

To determine if cell growth inhibition involved

cell cycle alterations, we examined the cell cycle

 phase distribution by flow cytometry of S180 cells.

After 24 h of CLI treatment, cells accumulated in G2/M

 phase (Fig. 3).

 Antitumor assay in vivo

As shown in Tables 2-4, after the ten-days of 

Fig. 2 The growth inhibitory effect of CLI on various tumor cells

Table 1 IC50

Value of CLI on different tumor cells (nM)

HeLa BEL-7402 HepG2 S180

48h 94.77 ± 0.12 38.56 ± 0.13 53.72 ± 0.13 -

72h 37.33 ± 0.13 17.70 ± 0.13 22.04 ± 0.13 2.83 ± 0.13

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Table 2 Inhibitory effect of CLI on H22 hepatocellular carcinoma in mice

GroupsDosage

(mg/kg)

Weight of mice (g)Tumor weight (g) Inhibitory rate (%)

Initiation Termination5 % GS - 20.3 ± 0.7 24.4 ± 0.9 3.08 ± 0.63 -

empty liposome - 20.4 ± 0.9 24.3 ± 0.7 2.90 ± 0.70 5.7

5-Fu 20 20.4 ± 0.9 21.9 ± 0.8 1.59 ± 0.79 ** 48.2

CLI (high) 0.11 19.9 ± 0.8 22.8 ± 0.6 1.68 ± 1.05 ** 45.2

CLI (medium) 0.055 20.4 ± 0.7 23.1 ± 0.5 1.83 ± 0.78 ** 40.4

CLI (low) 0.0275 20.5 ± 1.1 23.8 ± 0.8 2.30 ± 0.81 * 25.4

CLI (po) 0.11 20.7 ± 1.1 23.5 ± 0.8 2.06 ± 1.05 * 32.9

CLI (po) 0.055 20.9 ± 0.9 24.4 ± 0.9 2.41 ± 0.81 21.7

Each value is presented as the mean ± SD (n=10). Experiments were repeated three times. * P < 0.05, ** P < 0.01, compared with the

negative control.

Table 3 Inhibitory effect of CLI on S180 sarcoma in mice

GroupsDosage

(mg/kg)

Weight of mice (g)Tumor weight (g)

Inhibitory rate

(%)Initiation Termination

5 % GS - 21.1 ± 0.6 26.2 ± 0.5 3.11 ± 0.77 -

5-Fu 20 20.7 ± 0.8 22.7 ± 0.7 1.58 ± 0.65 ** 49.4

CLI (high) 0.11 21.0 ± 1.2 23.3 ± 1.1 1.85 ± 0.93 ** 40.7

CLI (medium) 0.055 20.6 ± 1.4 23.8 ± 1.4 1.90 ± 0.84 ** 38.9

CLI (low) 0.0275 20.5 ± 1.1 24.9 ± 1.3 2.29 ± 0.99 26.4

CLI (po) 0.11 20.5 ± 1.0 23.6 ± 0.9 2.17 ± 0.79 * 30.4

Each value is presented as the mean ± SD (n=10). Experiments were repeated three times. * P < 0.05, ** P < 0.01, compared with the

negative control.

Table 4 Inhibitory effect of CLI on Lewis lung carcinoma in C57BL/6 mice

GroupsDosage

(mg/kg)

Weight of mice (g)Tumor weight (g)

Inhibitory rate

(%)Initiation Termination

5 % GS - 20.8 ± 1.0 25.9 ± 0.9 3.18 ± 0.89 -

5-Fu 20 20.6 ± 0.8 21.8 ± 0.7 1.05 ± 0.39 ** 66.8

CLI (high) 0.11 21.0 ± 0.7 23.0 ± 1.0 1.66 ± 0.54 ** 47.6

CLI (medium) 0.055 20.7 ± 0.6 23.9 ± 0.8 1.81 ± 0.68 ** 43.2

CLI (low) 0.0275 20.5 ± 1.2 24.9 ± 0.7 2.52 ± 0.73 20.8

CLI (po) 0.11 21.2 ± 1.1 24.0 ± 0.9 2.18 ± 0.79 * 31.4

Each value is presented as the mean ± SD (n=10). Experiments were repeated three times. * P < 0.05, ** P < 0.01, compared with the

negative control.

treatments, CLI (0.0275, 0.55, 0.11  mg/kg)  and

5-fluorouracil (20 mg/kg) had significant inhibitory

effects on the growth of  transplanted tumors. The

r esults also indicate that, at the same doses (0.055

mg/kg, 0.11 mg/kg), the inhibitory rates due to  p.o. 

administration were lower than those due to i.v. 

administration. Empty liposomes had no inhibitory

effects on the growth of H22

cells. In addition,

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Fig. 3 Flow cytometric analysis of CLI- treated S180 cells

A: control; B: CLI 3 nM; C: CLI 5 nM

the average weight of CLI-treated mice was not

signicantly different than that of controls. However, 

5-fluorouracil (20 mg/kg) treatment inhibited normal

weight increase.

Cancer is one of the major causes of death

in the world and new useful treatments would

 produce significant improvements in public health. 

Common methods to cure cancer include surgery [6],

chemotherapy [7], radiotherapy [8], immunotherapy 

[9] and gene therapy [10]. All of these methods have

concomitant unwelcome side-effects. Finding new

effective cancer treatments has increasingly become

a primary task for scientists.  Published data have

confirmed that traditional Chinese medicines with

high activity and low toxicities might be developed

into the novel drugs for tumor therapeutics.Consequently, there has been growing interest in the

use of herbs as potent sources of new  therapeutic

anticancer drugs. Plants contain a wide variety

of chemicals that have potent   biological effects,

including anticancer activity [11]. The cucurbitacins

are of great interest because of the wide range of 

 biological activities that they exhibit.

The oral route for colloidal drug carrier systems

remains the most convenient and popular way of 

administration [12]. However, many anticancer drugsadministered orally can be eliminated from the rst-

 pass extraction by the cytochrome P450

-dependent

metabolic processes and the overexpression of 

 plasma membrane transporter P-glycoprotein (P-gp)

in the physiological systems involved (intestine,

liver, etc) [13].

The results of the present study suggest that CLI

signicantly inhibited the growth of tumor in vivo and

in vitro in a dose-dependent manner. Many anticancer 

agents arrest the cell cycle at the G1, S, or G2/M phaseand then induce apoptotic cell death [14, 15, 16]. In the

  present study, flowcytometric analysis showed that

CLI treatment arrested cells in G2/M phase. Therefore,

we presume that CLI may inhibit growth of S180

cells

via G2/M arrest. Further research on CLI is needed

in order to elucidate the exact mechanism of the

antitumor effect. In in vivo experiments presented,

CLI had strong antitumor activity even at relatively

low doses, while it had no significant effect on the

weight increase of the mice. At 0.055 mg/kg and0.11 mg/kg, CLI showed much higher efcacy by i.v. 

than by oral administration. In conclusion, our study

suggested that CLI has good perspective as a potent

and selective antitumor drug.

References

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