Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L₆)

Abstract

In order to provide a sensitive cell line model for investigating the mechanisms underlying the lymphatic metastasis of tumors and the effect of medicine against cells, a new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential (Hca-P/L₆) was established and the effect of curcumin on biological behavior of Hca-P/L₆ was observed. Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential (Hca-P) was subcutaneously inoculated into the medioventral line of a mouse 615 and the first generation of metastatic tumor cells of inguinal lymph node (Hca-P/L₁) was obtained. Then, Hca-P/L₁ was screened by the route of mouse foot pad subcutaneously → lymph node → scale-up culture in vitro → mouse foot pad subcutaneously for five times consecutively. The sensitivity of two murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L₆) and two anchorage-dependent human hepatocarcinoma cell lines (SMC7721 and HepG₂) to curcumin were studied by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after these cells had been pretreated by curcumin at the concentration of 15-240 μmol/L for 48 h. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 μmol/L in vitro, the effect of curcumin against cell proliferation of Hca-P and Hca-P/L₆ was observed by inverted microscope, cell growth curve and cell population doubling time; the effects of curcumin on cell cycles of Hca-P/L₆ and Hca-P were studied by flow cytometry (FCM). The results showed Hca-P/L₆ spreading to the lymph nodes at multiple sites in mice was screened from Hca-P. The lymph node metastatic rate was 100%. Curcumin had significant growth inhibiting effect on both murine ascites and human hepatocarcinoma cell lines in a dose-dependent manner (P<0. 05). At concentrations of 30-120 μmol/L, curcumin had more inhibition on murine ascites hepatocarcinoma cell lines than on human anchorage-dependent hepatocarcinoma cell lines. At concentrations of 60-240 μmol/L, curcumin had more inhibition on Hca-P/L₆ with (the 50% inhibitory concentration) IC₅₀ of 51.48 μmol/L than on Hca-P with IC₅₀ of 90.87 μmol/L. After pretreatment by curcumin at the maximum non-cytotoxic dose of 15 mol/L for 7 days, the proliferations of Hca-P/L₆ and Hca-P were inhibited (P<0.05) in a time-dependent manner (P<0.01) and the population doubling time of Hca-P/L₆ and Hca-P was prolonged (P<0.01), and curcumin had more inhibition on Hca-P/L₆ than on Hca-P (P<0.05). After pretreatment by 15 μmol/L curcumin for 48 h, the morphous of Hca-P/L₆ was influenced more seriously than that of Hca-P and the cell cycle was redistributed with Hca-P/L₆ being blocked in the S phase and Hca-P in the S and G₂/M phases. Hca-P/L₆ was validated to be more sensitive to curcumin than Hca-P. Hca-P/L₆ is a novel sensitive cell line model for investigating the mechanisms underlying tumor lymphatic metastasis and the effect of the medicine against cells.

Keywords

murine ascites hepatocarcinoma cell line / metastasis / curcumin / drug sensitivity

Introduction

The study on effective substance of traditional Chinese medicines (TCMs) is the key point of quality control on TCMs and the discovery and development of new drugs of natural origin. It is a potentially effective method to study bioactive compounds in TCMs to screen and separate bioactive compounds in TCMs under the condition of retaining the situ, integrity and activity of cells, cell membrane and receptor, and moreover reflecting which compounds interact with which targets and how much their affinity is. However, the reactivity of a variety of classes of cells to the same kind of medicine is different and the sensitivity of variant clone cell strains from the same cell line to the same kind of medicine is also different. Zhang et al. screened the potential bioactive components in the combined prescription of Danggui Buxue (当归补血) decoction by a cell extraction coupled with high-performance liquid chromatography (HPLC) analysis, and the results show the number of compounds detected from HL-7702 cell, RAW 264.7 cell and Caco-2 cell extractions were 9, 7 and 13 respectively [1]. Therefore, it is necessary to select a corresponding cell strain when the effects of the medications on the specialized tissues or cells are investigated. For this reason, it is very important to apply a more sensitive cell model to screen bioactive components in TCMs. At present, the Caco-2 cells, human adenocarcinoma cells, have been extensively used as a relevant physiological model for investigations of screening and developing new drugs, intestinal absorption mechanism of medications and as a metabolism model in vitro [2-5]. In 1990, two cell strains with different lymphatic metastatic potentials were separated from a murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential, Hca-F25/L by Ling et al. [6], of which the lymphatic metastasis rate of high metastatic clone strain Hca-F (16A_3-F_3) was 78.6%-89.4%, and that of low metastatic clone strain Hca-P (A_2-0), which was 15%-15.4%, and had been widely used in studying the mechanism underlying tumor lymphatic metastasis and screening drugs [7-10].

According to the tumor heterogeneity theory, different subclone cell strains of a malignant tumor may present a difference in growth rate, invasion ability, reaction to growth signal and sensitivity to anticancer medicines [11]. In 1992, Chen et al. [12] studied the stability of highly metastatic clone cells 16A_3 and lowly metastatic clone cells A_2_0 from murine ascites hepatocarcinoma, and the results suggested that the effective maintaining time of the highly metastatic clone cells 16A_3 was within 20 passages. In 2003, Bai et al. [13] cloned murine ascites hepatocarcinoma cell strain with high lymphatic metastatic potential Hca-F again, and obtained three cell strains with different lymphatic metastatic potentials. Therefore, it is essential to screen a pair of murine ascites hepatocarcinoma cell lines with different lymphatic metastatic potentials from lowly metastatic cells for offering two reciprocally compared tumor cells to study the mechanism underlying tumor lymphatic metastasis and screening bioactive compounds from TCMs. In this paper, murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential (Hca-P) was used as source tumor to screen a novel murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential through the lymphatics in mice. Curcumin, a kind of TCM with anticancer property, was used as the experimental medicine to determine the sensitivity of two murine ascites hepatocarcinoma cell lines to curcumin in comparison with two human anchorage-dependent hepatocarcinoma cell lines (SMC7721 and HepG₂) by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, the effects of curcumin on the biological behavior of these two murine ascites hepatocarcinoma cell lines were observed by a cell growth curve, cell population doubling time and flow cytometry (FCM), and it was evaluated if there was different sensitivity to cucumin between these two murine ascites hepatocarcinoma cell lines. The results may provide a potential cell line available for the study of the mechanism underlying tumor lymphatic metastasis and screening bioactive compounds from TCMs, and offer some experimental proofs for the therapeutic potential of curcumin against the lymphatic metastasis of malignant tumors.

Materials and methods

Chemicals and materials

Chemicals

Curcumin of analytical grade, molecular formula C₂₁H₂₀O₆, molecular mass 368.39, was purchased from the Third Shanghai Reagent Plant (China). MTT and Dimethylsulfoxide (DMSO) were from Sigma-Aldrich (USA). RPMI 1640 was procured from Gibco BRL (NY, USA). New born bovine serum (NBBS) was from the Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (China). Isotonic sodium chloride of 0.9% was from the Otsuka Pharmaceutical Co., Ltd. (Japan).

Equipment

The water-jacketed CO₂ incubator was a product of NAPCO (USA). The superclean bench was from Suzhou Antai Air Tech Co., Ltd. (China). The LDZ5-2 automatic equilibrium centrifugal machine was purchased from Beijing Medical Centrifuge Factory (China). The MP2002 electronic balance was from the Shanghai Hengping Scientific Instrument Co., Ltd (China). The ELISA Reader MultiskanMk3 was from Finland Labsystem (Finland). The EP ICS XL flow cytometer was from Coulter (USA). The inverted microscope was from Olympus Optical Co., Ltd. (Japan), 96-well and 24-well microplates were from Millipore Co. (USA), and the germ free membrane & syringer filters were from Costar (USA).

Cell line

Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential (Hca-P) was kindly provided by Prof. Jianwu TANG from the Pathology Department of Dalian Medical University (China). Human hepatocarcinoma cell lines (SMC7721 and HepG₂) were purchased from the Shanghai Cell Institute of the Chinese Academy (China).

Experimental animal

Male mice 615, aged 6-8 weeks, weighing 18-22 g, were provided by Experimental Animal Center of Dalian Medical University (China).

Methods

Preparation of solutions

Curcumin solution was prepared as follows: curcumin powder of 7.4 mg was accurately weighed by an electronic scale and dissolved in 1 mol/L NaOH to adjust pH value to 7.2. Then, 20 mL RPMI 1640 medium supplemented with 10% NBBS was added to get a stock curcumin solution at a concentration of 1 mmol/L. The stock solution was filtered for degerming and stored away from light at 4°C within two weeks. Preparation of MTT solution is as follows: MTT powder was accurately weighed and dissolved in 0.01 mol phosphate buffered solution (PBS, pH 7.4) to reach a concentration of 5 mg/mL. The solution was filtered for degerming and stored away from light at 4°C within two weeks for effective use.

Cell culture

Hca-P cells were defrosted quickly and washed with 9% isotonic sodium chloride twice. The cell suspension (0.2 mL, about 6×10⁶ cells) was intraperitoneally injected into a mouse 615 to incubate in duplicate for two passages in order to harvest a greater amount of cells in less than 2 weeks. The cells in ascites were extracted and seeded into culture bottles at a density of 1×10⁵ cells/mL in RPMI 1640 medium supplemented with 10% NBBS and 8000U gentamycin sulfate injection (Yantai Justaware Pharmaceutical Co., Ltd., China) with pH value of 7.2 at 37°C in a 5% CO₂ atmosphere for 48 h. Human hepatocarcinoma cell lines were defrosted and maintained in culture bottles and then collected by enzymatic digestion to prepare for the experiment when the cells reached 80% confluence.

Obtaining of the first generation of metastatic tumor cells in a lymph node of mouse 615 from Hca-P and screening for the cells in mouse lymphatics

Cell suspension (0.1 mL, about 2×10⁶ cells) of the second passage of ascites Hca-P cells of mouse 615 was subcutaneously inoculated into the medioventral line of a mouse 615. After 21 days, inguinal lymph nodes of the mouse were taken out. Half of a lymph node was kept for pathological examination and the other half was made into a cell suspension. The cells were seeded into culture solution for expanding culture. The first generation of metastatic tumor cells of lymph node of mouse 615 was named as Hca-P/L₁. Exponentially growing Hca-P/L₁ cells were washed and diluted with cold isotonic soddium chloride to reach a density of 2×10⁷ cells/mL, and 0.05 mL of such cell suspension (1×10⁶ cells) was subcutaneously inoculated to the right foot pad of every mouse 615 at two mice per time. Metastatic tumor of a lymph node → scale-up culture in vitro → metastatic tumor of a lymph node, repeated passages were made for five times. The second generation of metastatic tumor cells of a lymph node of mouse 615 from Hca-P was named as Hca-P/L₂. By analogy, the sixth generation of metastatic tumor cells was named as Hca-P/L₆.

Mensuration of lymph node metastatic rate in mice 615 induced by Hca-P and Hca-P/L₆

Forty male mice 615 were assigned to two groups (20 mice of each group): Hca-P group and the sixth generation of lymph node metastatic tumor cell (Hca-P/L₆) group. Tumor cell suspension of 0.05 mL (1×10⁶ cells) was subcutaneously inoculated into the right foot pad of each mouse 615. The tumor appearance time and the state of tumor were visually observed by the naked eyes,an the health status and the death time of the mice were recorded. When a mouse was found dead, the dissection was performed at once with the transplanted tumor in situ in the inoculating site of the foot pad of the mouse. The lymph nodes of popliteal, inguinal, axillary at both sides, submaxillary, celiac and viscera lymph nodes as well as liver, lung, kidney and brain were taken out and fixed in 10% formaldehyde solution. The tissues were examined by pathology, and the lymph node metastasis or organ blood metastasis was studied microscopically.

Cytotoxicity of curcumin in cells detected by MTT assay

The in vitro cytotoxicity of curcumin against murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L₆) and human anchorage-dependent hepatocarcinoma cell lines (SMC7721 and HepG₂) were evaluated and compared by using the MTT assay, and the trypan blue dye exclusion assay was used to assess cell viability as previously described [14]. The above cell lines, at the exponential growth phase, were washed and seeded in 96-well microtiter plates at a density of 1×10⁵ cells/mL for 100 μL/well. Each well contained 10⁴ cells. Standard curcumin was suspended in 1 mol/L NaOH and dilution series were prepared in an RPMI medium with 10% NBBS and 100 μL of each dilution step was added in each plate well to obtain six concentrations of curcumin ranging from 0 to 240 μmol/L in triplicate (concentrations of curcumin: 240, 120, 60, 30, 15 μmol/L and 0). Control cells received only the vehicle. The plates were incubated at 37°C in a 90% humidity atmosphere of 5% CO₂ for 48 h, and then MTT, diluted in 1640 RPMI medium at a concentration of 5 mg/mL (20 μL/well), was added. Following an incubation period of 4 h, the cell culture medium was discarded and each well was added with 200 μL DMSO and shaken for 5 min to dissolve the formazan derivatives. Measurement of the intensity was carried out spectrophotometrically by an ELISA reader at a wavelength of 570 nm. Finally, the cytotoxicity of curcumin for each cell line was calculated based on its effect on the color yield of treated cells following MTT exposure. It is defined as the drug concentration that induced a 50% reduction in control absorbance (A) value, i.e. the toxicity of samples was expressed as the drug concentration at which the drug could inhibit the growth of the cells by 50% (IC₅₀) compared with control. To determine the IC₅₀, the percent of control, the A value was plotted for each concentration (mean of the 9 wells) against the log [drug] using Office 2000 Excel software. Each experiment was repeated at least 3 times.

The percentage of cell viability was calculated as follow: cell viability (%)=(A_sample/A_control)×100% [A_sample is the A of cells tested with various formulations, A_control is the A of control cells (incubated with cell culture media only)]

The percentage of cell growth inhibition was calculated as follows: cell growth inhibition (%)=1-cell viability (%)

Cell growth curves and population doubling time of murine ascites hepatocarcinoma cell lines

Two murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L₆) at the exponential growth phase were washed and seeded in 24-well microtiter plates at a density of 1×10⁴ cells for 1 mL/well. The drug group and drug-free control group were assigned. The drug group contained curcumin at the maximum non-cytotoxic concentration prepared in RPMI medium with 10% NBBS, and the control group received only the vehicle. The plates were incubated at 37°C in a 5% CO₂ at 90% humidity atmosphere for 7 days. The cell number was examined every 24 h from day 2 to day 7. At the indicated time points, cells were detached from the plates, and the cell number was counted under a microscope using a hemocytometer. Each experiment was performed in a triplicate set-up and then the cell growth curve was drawn. According to the Patterson Formula, the cell population doubling time at the exponential growth phase was calculated: Td=T^{log2/log(Nt/N}₀⁾ (Td means population doubling time (h); T means the time length for the cell number increased from N0 to Nt; N means cell number).

Cell cycle progression of murine ascites hepatocarcinoma cell lines by flow cytometry

Two murine ascites hepatocarcinoma cell lines (Hca-P and Hca-P/L₆) were respectively seeded in 100 mL-culture flasks at a density of 4×10⁵ cells/mL for 20 mL/flask overnight and synchronized by incubating in media supplemented with 0.1% NBBS for 48 h in incubator at 37°C. After serum starvation, the drug group and drug-free control group were assigned. The drug group contained curcumin at the maximum non-cytotoxic concentration prepared in RPMI medium with 10% NBBS, and control group received only the vehicle. Cells in culture media containing 10% NBBS were cultured for 48 h at 37°C in incubator. Subsequently, non-cytotoxic cells were washed with cold PBS and pelleted by centrifugation at 500 r/min for 5 min at 4°C for twice. The cells were then fixed with ice-cold absolute ethanol (2 mL/flask) at -20°C overnight. For staining, fixed cells were centrifuged at 500 r/min for 5 min at 4°C. After the removal of ethanol, the cells were re-suspended in 500 μL DNA staining solution containing 8 μg/mL RNase A and 40 μg/mL propidium iodine and incubated for 30 min at 4°C. The stained cells were analyzed for fluorescence intensity with a fluorescence-activated cell sorter equipped with an argon laser at an emitting wavelength of 488 nm, using the CellQuest software. The percentages of cells in G₀/G₁, S and G₂/M cell cycle phases were calculated by the Modfit 2.0 software.

Statistical analysis

Statistical analysis was performed by SPSS 12.0 statistical software. Data was described as the \overline{\mathrm{x}}±s, and analyzed by Student’s t-test. The chi-square test was used to compare treated and control groups and treated groups among one another. Significance of differences was set at P<0.05.

Results

Morphological characteristics of Hca-P/L₁ and Hca-P/L₆ and their lymph node metastatic rates

After the Hca-P cells were inoculated subcutaneously into the medioventral line of the mouse 615 for 21 days, the right inguinal lymph node of the mouse was swollen and a locally transplanted tumor in the medioventral line of the mouse at the inoculating site was found, which was white-grayish and hard in texture. The histology of the transplanted tumor is shown in Figure 1. The tumor was a poorly differentiated lesion, which was made up of multilateral tumor cells, which varied in size with some large multinucleate anaplastic tumor giant cells. Local necrosis of the tumor cells was observed. Lamellar necrosis was detected in a part of the tumor cells and interstitial blood vessels were sparse. The histology of the swollen right inguinal lymph node (Hca-P/L₁) can be seen in Figure 2, where the normal structure of the lymph node was preserved partially and large multilateral tumor cells were distributed in the lamellar area which had similar morphology with the transplanted tumor cells, indicating that the tumor tissue of this lymph node was the metastatic one from the transplanted tumor. Hca-P/L₁ were cultured in medium and their morphology is shown in Figure 3. The tumor cells grew in suspension in a large round shape and had clear cytoplasm. Some of them were in clusters and a few large cells were in the state of differentiation and proliferation. Their volume was bigger than that of Hca-P (Fig. 4). At the beginning of the culturing, ascites tumor cells were relatively in the same size. But, if culturing time was excessive, cytoplasmic granulation became evident and the cell outline was enhanced. It suggested that the cell culture time should not be too long, instead, cells were in the optimal living state when cultured for 2-4 days. If the culture time was too long, the cells would decay.

When Hca-P/L₁ were repeatedly inoculated 5 times to passage in the lymphatics of mice, lymph node metastasis would occur, at the latest, 16 days later with a 100% metastatic rate. During the progress of the 5 passages of Hca-P/L₁-Hca-P/L₅, every transplanted tumor at the mouse pad grew rapidly. The extremity inoculated by tumor cells was incomplete and necrotic, and the tumor formed at the extremity root. With the increasing passages, the inguinal and axillary nodes were enlarged at increasing speed.

Comparison of lymph node metastatic rates in mice 615 induced by Hca-P and Hca-P/L₆

Hca-P and Hca-P/L₆ cells were subcutaneously inoculated into the right foot pad of 20 mouse 615, respectively (Table 1). The lymph node metastasis rate from Hca-P is 20% with only inguinal and axillary node metastasis and that from Hca-P/L₆ is 100%, spreading widely to multiple sites all over the body. The number of mice with popliteal, inguinal, axillary, celiac and renal hilar lymph node metastatic tumors was 16, 14, 15, 5 and 2, respectively. The tumors spread simultaneously to popliteal and inguinal, popliteal and axillary, inguinal and axillary lymph nodes in 13 mice, respectively. Some tumors spread simultaneously to popliteal, inguinal and popliteal lymph nodes in 12 mice. Some tumors spread simultaneously to popliteal, inguinal, popliteal and celiac lymph nodes in 5 mice, of which 2 mice had renal hilar lymph node metastasis.

After Hca-P/L₆ cells were inoculated subcutaneously into the right pad of mouse 615 for 21 days, a white tumor was found in the right renal hilus of 2 mice. The tumor in each mouse was linked with the adrenal gland and coated within the renal capsule. The tumor along with the renal capsule was taken out and examined histologically. The tumor close to the kidney from the first mouse was composed of large undifferentiated cells multilateral in shape, varied in size, these tumor cells were histologically similar to the cells of the transplanted tumor from the mouse 615 inoculated with Hca-P/L₅, and they were metastatic tumor cells from the transplanted tumor of Hca-P/L₅. The tumor was separated from the kidney by a thick fibrous tissue where the tumor cells infiltrated, but there were no tumor cells in the renal tissue. Figure 5 shows the relationship of the tumor close to the kidney with the adrenal gland in the first mouse. The tumor was adjacent but not invasive to the adrenal gland. The adrenal gland capsule was intact and there was no tumor tissue in the adrenal gland. It suggested that this metastatic tumor was neither in the renal parenchyma nor in the adrenal gland parenchyma, that is to say, it was not a hematogenous metastatic tumor. The relationship of the tumor close to the kidney with the kidney and its adrenal gland in the second mouse was similar to the findings in the first mouse. Figure 6 presents the histology of the tumor close to the kidney in the second mouse. Lymphatic tissue was found in the tumor. The tumor was made up of large, undifferentiated, multilateral cells varied in size and scattered in the lymphatic tissue, indicating that the source tissue of this tumor might be a lymph node. This tumor was from a lymphatic spread instead of a hematogenous spread. The liver, lung, spleen and brain tissues were examined pathologically and no metastatic tumor tissue was found which indicated that no hematogenous spread occurred in Hca-P/L₆.

Inhibition of curcumin on Hca-P/L₆, Hca-P, SMC7721 and HepG₂in vitro

The effect of curcumin on cell viability was assessed by using MTT assay, and the results are shown in Table 2 and Figure 7. Growing culture cells of Hca-P/L₆, Hca-P, SMC7721 and HepG₂ were treated with curcumin at concentrations ranging from 15 to 240 μmol/L for 48 h, and the proliferation of all cell lines were markedly inhibited. The inhibition effect of curcumin on two human hepatocarcinoma cell lines was not as stable as its inhibition effect on the two murine ascites ones. As shown in Figure 7, at the concentrations of 30, 60 and 120 μmol/L, the inhibition of curcumin on Hca-P/L₆ and Hca-P was stronger than that on SMC7721 and HepG₂ (P<0.05). The ascites hepatocarcinoma cell lines were more sensitive to curcumin than human hepatocarcinoma cell lines. Through analysis with the chi-square test, the inhibition rates of curcumin at 5 concentrations against Hca-P and Hca-P/L₆ cells were significantly different (P<0.05) in a dose-dependent manner. Through the t test, the inhibition of curcumin at each identical concentration (the concentrations ranging from 60 to 240 μmol/L) on Hca-P/L₆ was stronger than that on Hca-P, and the scope of its inhibition on Hca-P/L₆ was broader than that on Hca-P. When curcumin was at 15 μmol/L, the cell survival rates of Hca-P/L₆ and Hca-P were both more than 90%, and this concentration was the maximal non-cytotoxic concentration or minimal effective concentration. With dose-response relationship, IC₅₀ of cell viability was calculated. The IC₅₀ of Hca-P/L₆ and Hca-P was 51.48 and 90.87 μmol/L, respectively, indicating that Hca-P/L₆ were more sensitive to curcumin than Hca-P.

Effects of curcumin on cell growth curves and population doubling time of Hca-P/L₆ and Hca-P

The effects of curcumin on cell growth curves of two cell lines are shown in Figure 8. In both the drug group and control group, 1×10⁴ cells were seeded simultaneously and observed for successive 7 days, and the results show that 15 μmol/L curcumin could inhibit the proliferation of Hca-P/L₆ and Hca-P (P<0.05, n=18). Moreover, the longer it took the time, the stronger the inhibitory effect was (P<0.01). Through comparison of the variation of cell inhibitory rate, it was found that the inhibition effect of curcumin on Hca-P/L₆ was stronger than that on Hca-P (P<0.05, n=18). In drug group or control group, cell proliferation rates of Hca-P/L₆ and Hca-P were significantly different, and the growth velocity of Hca-P/L₆ was faster than that of Hca-P (P<0.05).

The effect of curcumin on the population doubling time (PDT) of the two cell lines is shown in Table 3. The results show that the PDT of the Hca-P cell was significantly longer than that of the Hca-P/L₆ cell (P<0.05). After treatment with curcumin, the PDT of the two cells lines became longer (P<0.01), indicating that curcumin could inhibit the proliferations of the two cells lines. But in the sets of test cells, the PDT of the Hca-P/L₆ cells are not significantly different from that of Hca-P cells (P>0.05), which indicates that the inhibition of curcumin on Hca-P/L₆ is stronger than that on Hca-P.

Effect of curcumin on cell cycles of Hca-P/L₆ and Hca-P

After treatment with 15 μmol/L curcumin for 48 h, the cell cycle distributions of Hca-P/L₆ and Hca-P by flow cytometry are shown in Fig. 9, and the analytic results of the distribution of DNA content of the gated cell population is shown in Table 4, suggesting that cell cycle distributions of Hca-P/L₆ and Hca-P were different without curcumin treatment, and the percentage of Hca-P/L₆ in G₁/G₀ phase was significantly lower, and the percentage in G₂/M phase was higher than that of Hca-P, which indicated that the proliferation of Hca-P/L₆ was more active than Hca-P. After treatment with curcumin, the cell cycle distributions of the two cell lines were also different. The percentage of Hca-P/L₆ in the G₁/G₀ phase was significantly lower, and the percentage in the S phase was higher than that of Hca-P (percentage of Hca-P/L₆ cell in G₂/M phase: 0; percentage of Hca-P cell in G₂/M phase: 6.43%). Through analysis of the chi-square test, the cell cycle distributions of the two cell lines were significantly different between the curcumin group and the curcumin-free group (P<0.05). Curcumin could change the cell cycle distribution by blocking Hca-P/L₆ in the S phase, promoting Hca-P from G₁/G₀ phase to G₂/M phase, which indicated that the effects of curcumin on cell cycle progressions of the two cell lines were different, and the effect of curcumin on blocking Hca-P/L₆ was stronger.

Discussion

Murine ascites hepatocarcinoma cell line H_(22) belongs to the mouse liver carcinoma which is a solid liver tumor of C3H mice induced by smearing o-aminoazotoluene (OAAT) on the ears of C3H mice for 100 times by the Academy of Medical Science of the Soviet Union in 1952 and passaged subcutaneously. This solid tumor was introduced into China in 1959, and was transformed into the ascites tumor by the Shanghai Institute of Pharmaceutical Research, Chinese Academy of Sciences in 1963 (the ascites type was also called Hca, HAC or HepA), and gained passages in the abdominal cavities of Km mice. H_(22) tumor subcutaneously inoculated into the mice and grew into a spherical nodule form. The tumor inoculated into the mice abdominal cavity proliferated in suspension. Due to the difference of mice strains and lymphatic metastatic potentials, 80320 cell line [15], H22-F0/L cell line [16] and H22-F25/L cell line [17] were established in vitro. The transplantation rates of these three cell lines were 100%, but their metastatic properties were different. Five cloning hepatocarcinoma cell strains with different lymphatic metastatic potentials were separated from Hca-F25/L cell line in 1990 and they all spread in the lymphatics. All these 5 clone strains and their parental generation retained the characteristics of epithelial tissue. By chromosome analysis, every clone strain had 4 marker chromosomes which are the same as its parental generation, indicating that they had genetic relationship and demonstrating that the Hca cell line was heterogeneous in phenotype and karyotype [6]. Hca-F and Hca-P have been used for many years and these two cell strains might produce their own subclones different in properties such as metastatic potential. Therefore, it is necessary and available to screen novel stable murine ascites hepatocarcinoma cell lines with low and high lymphatic metastatic potential.

In this paper, murine ascites hepatocarcinoma cells with low metastatic potential Hca-P was subcutaneously inoculated to the medioventral line of a mouse 615 to produce the first generation of metastatic tumor cells of lymph node (Hca-P/L₁). The lymph node metastatic tumor cells still possessed the characteristics of their parental generations Hca-P and Hca cells, growing in suspension [16, 17]. Via the pad subcutaneously → metastatic tumor of lymph node → scale-up culture in vitro → the pad subcutaneously, 5 consecutive passages were repeated, the murine ascites hepatocarcinoma cells, with high metastatic potential and 100% lymph node metastasis rate, Hca-P/L₆ was screened. The metastasis rate of Hca-P/L₆ was higher than that of the past high lymphatic metastatic tumor cell strain, with multi-metastatic sites and stable metastatic property. The second generation of metastatic tumor cells of lymph node of mouse 615 from Hca-P was marked as Hca-P/L₂. By analogy, the sixth generation of metastatic tumor cells of lymph node of mouse 615 from Hca-P was Hca-P/L₆. This cell line was similar to Hca-P in genetic background but different significantly from Hca-P in metastatic phenotype. Different from the method screening a high metastatic tumor cell line from a high metastatic tumor cell line, the present method screens a high metastatic tumor cell line from a low metastatic tumor cell line. Different from the screening mode by abdominal cavity → the pad → lymph node → abdominal cavity, the present mode was to use scale-up the culture of lymph node metastatic tumor cells in vitro instead of the abdominal cavity. The result shows that the metastasis occurred earlier and the time of model screening was shortened.

Culture cells in vitro are divided into two categories: anchorage-dependent culture and suspension culture according to whether the cells required a solid substratum for growth, i.e., the solid glass or plastic surface of a culture dish or micro-carrier beads, or not. Anchorage-dependent cells require a solid growth substrate for growth. Most culture cells deriving from solid animal tissues are anchorage-dependent. Trypsin must be used to detach and separate anchorage-dependent cells from the surface of the solid growth substrate. If the reaction of trypsin goes beyond the limit or fails to do well, the trypsin can damage the structure of cell membrane and affect the experimental results. Suspension culture cells do not require a solid growth substrate for growth, but grow in a suspension manner. Suspension culture offers advantages over two-dimensional, monolayer culture and can overcome the limitation of cell growth area and the lack of homogeneity in two-dimensional culture systems. These advantages make suspension culture easy to proliferate for the production of large number of cells. When they are treated with medicine, suspension culture cells can contact thoroughly and sensitively with the medicine. Moreover, they do not need a digestive reagent and the cell membrane will not be damaged. Hca-P/L₆ can be passed in the mouse abdominal cavity and can grow freely in suspension. It is easy to harvest large number of cells with very good activity in a short time, and thus can be applied to screen medicine in large scales.

Curcumin, a kind of polyphenolic phytochemical derived from the perennial herb Rhizoma Curcumae Longae, has effects of anti-inflammatory, anti-oxygen and anti-tumor natures, and is one of the pure chemical components of TCMs which have been studied most extensively and completely. Many researches have shown that curcumin can inhibit the proliferation of many tumor cell lines in vitro and has the possibility to inhibit invasion and metastasis of tumors [18-21]. MTT assay results indicated that the ascites hepatocarcinoma cell lines were more sensitive to curcumin than human anchorage-dependent hepatocarcinoma cell lines, and high lymphatic metastatic Hca-P/L₆ was more sensitive to curcumin than its parental cell strain Hca-P. The sensitivity of curcumin to anchorage-dependent culture cells was different from that to suspension culture cells, and the difference of tumor cell lines led to the difference of sensitivity to curcumin. The tumor cell population of the same cell line was not homogeneous, and the sensitivity of subclones to the medicine was different. The mechanisms may be that tumor cells with high metastatic potential having a higher proportion of cells in the exponential growth phase than those with low metastatic potential, and antitumor drugs usually exert its effect on the cells in mitotic phase. The mechanisms may be concerned with curcumin controlling the expression and regulation of tumor differentiated genes.

Without curcumin treatment, Hca-P/L₆ and Hca-P all grew in suspension, and the division growth was active. Hca-P/L₆ is significantly different from Hca-P in cell growth rate, cell population doubling time and cell cycle distribution. The above-mentioned indicated that the lymphatic metastatic potential of the two cell lines was different and the reproductive activity difference was also obvious. The results from the MTT assay, cell growth curve, cell population doubling time and cell cycle distribution demonstrated that curcumin could inhibit the cell proliferation of Hca-P/L₆ and Hca-P. The mouse ascites hepatocarcinoma cell lines were more sensitive to curcumin than human hepatocarcinoma cell lines and could be taken as the target cells to research the effect of TCMs with the characteristics of “multi-component, target, and channel” on the cells. But the results from the analyses of cytotoxicity, cell growth curve, cell population doubling time and cell cycle distribution had shown that the inhibition of curcumin on Hca-P/L₆ was stronger than that on Hca-P, and curcumin could inhibit the cell proliferation of Hca-P/L₆ and Hca-P. Hca-P/L₆ was more sensitive to curcumin than Hca-P. Hca-P/L₆, with high metastatic potential, originated from Hca-P. There was heterogeneity between Hca-P/L₆ and Hca-P. The different biological characteristics between the two subclones led to different sensitivities to the same medicine. This research result can provide a comparatively ideal cell model for further studying the molecular mechanisms underlying the effect of medicine against tumor cells.

References

[1] ZhangX,QiL W,YiL,LiP,WenX D,YuQ T.Screening and identification of potential bioactive components in a combined prescription of Danggui Buxue decoction using cell extraction coupled with high performance liquid chromatography.Biomed Chromatogr, 2008, 22(2): 157-163

[2] DantzigA H,DuckworthD C,TabasL B. Transport mechanisms responsible for the absorption of loracarbef, cefixime and cefuroxime axetil into human intestinal Caco-2 cells. Biochem Biophys Acta, 1994, 1191(1): 7-13

[3] HidalgoI J,RaubT J,BorchardtR T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology, 1989, 96(3): 736-749

[4] MeunierV,BourrieM,BergerY. The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications. Cell Biol Toxicol, 1995, 11(3,4): 187-194

[5] ArturssonP,KarlssonJ. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem Biophys Res Commun, 1991, 175(3): 880-885

[6] LingM Y,LiuX F,GuanS Q,Yang, LongX,ZhengH Z,GuS Z,YuQ N. The screening and characters of cell clone with different metastatic ability. Dalian Yi Ke Da Xue Xue Bao, 1990, 1: 1-7 (in Chinese)

[7] LiuM,ZhuJ,YangP M,ZhangJ N. The expression of L-selectin in mouse liver tumor cell HCa-F and HCa-P and the potential relationship with metastasis. Jie Pou Xue Jin Zhan, 2003, 2: 100-104 (in Chinese)

[8] GuoL Y,ShiG X,GaoZ H,ShenJ,XingR,QianZ C. Response to hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 from elemene combo tumor cell vaccine. Cell Mol Immunol, 2006, 4: 291-295

[9] SunC R,TangJ W,SunM Z,LiuS Q,ZangH Y,WangB,SongB,ZhangY N,ZhangZ Q,ZhaoZ Y. Identification lymphatic metastasis-associated proteins in mouse hepatocarcinoma cell Lines using quantitative proteomics technique. Sheng Wu Hua Xue Yu Wu Li Jin Zhan, 2007, 8: 856-864 (in Chinese)

[10] XieC S,GaoW C,ChenP Fg,WuL C. The effect of Bcl-2 and bax of Chinese medicine ATU-I on 615 mice with HCa-F liver carcinomas. Zhong Hua Zhong Yi Yao Xue Kan, 2007, 1: 2327-2329 (in Chinese)

[11] FidlerI L. Tumor heterogeneity and biology of cancer invasion and metastasis. Cancer Res, 1978, 38(9): 2651-2660

[12] ChenS,LingM Y,LiuXF,ZhangC H. The stability of highly and lowly metastatic clonal cells from murine ascites hepatocarcinoma. Dalian Yi Ke Da Xue Xue Bao, 1992, 14: 32-37 (in Chinese)

[13] BaiJ,WangB,HouL,LiY,LingM Y,LvS,BaiY G.A fresh clone of small rat of ascites type liver cancer high lymph path transfer tumor individual plant HCa-F. Dalian Yi Ke Da Xue Xue Bao, 2003, 25(1): 65-66 (in Chinese)

[14] BrombergN,JustoG Z,HaunM,DuranN,FerreiraC V. Violacein cytotoxicity on human blood lymphocytes and effect on phosphatases. J Enzyme Inhib Med Chem, 2005, 20: 449-454

[15] LiuD F,ZhangB D,SunY L. The establishment of hepatic carcinoma cultured cell strain 80320 of mice. Ai Zheng, 1984, 3(3): 186-188, 230 (in Chinese)

[16] LingM Y,ZhangC,GaoW S,ZhuF C. Establishment of experimental lymphatic metastasis model of transplanting murine ascites hepatocarcinoma (H(22)). Dalian Yi Ke Da Xue Xue Bao, 1983, 2: 9-13 (in Chinese)

[17] LuW D,YangJ.Observation of the growth methods and general biological characteristics of murine ascites hepatocarcinoma (Hca). Zhong Guo Zhong Liu Lin Chuang, 1979, 3: 188-192 (in Chinese)

[18] LingM Y,WangM Y,ZhangC,LiuX F. An observation on the morphology and metastatic potential to the ascites hepatomas (H(22)) in mice. Dalian Yi Ke Da Xue Xue Bao, 1987, 1: 27-32 (in Chinese)

[19] ShaoZ M,ShenZ Z,LiuC H,SartippourM R,GoV L,HeberD,NguyenM. Curcumin exerts multiple suppressive effects on human breast carcinoma cells. Int J Cancer, 2002, 98(2): 234-240

[20] MenonL G,KuttanR,KuttanG. Inhibition of lung metastasis in mice induced by B16F10 melanoma cells by polyphenolic compounds. Cancer Lett, 1995, 55(1-2): 221-225

[21] KuttanR,BhanumathyP,NirmalaK,GeorgeM C. Potential anticancer activity of turmeric (Curcuma longa). J Cancer Lett, 1985, 29(2): 197-202

[22] KhafifA,SchantzS P,ChouT C,EdelsteinD,SacksP G. Quantitation of chemopreventive synergism between epigallocatechin-3-gallate and Curcumin in normal, premalignant and malignant human oral epithelial cells. Carcinogenesis, 1998, 19(3): 419-424

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