Tian Xian Liquid (TXL) induces apoptosis in HT-29 colon cancer cell in vitro and inhibits tumor growth in vivo

Background Tian Xian Liquid (TXL) is a Chinese medicine decoction and has been used as an anticancer dietary supplement. The present study aims to investigate the effects of TXL on the apoptosis of HT-29 cells and tumor growth in vivo. Method HT-29 colon cancer cells were treated with gradient dilution of TXL. The mitochondrial membrane potential was measured by JC-1 assay. The release of cytochrome c from mitochondrial and apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3, 9 were examined by Western blot analysis. HT-29 cells were implanted in nude mice to examine the effects of TXL on tumor growth. Result TXL inhibited HT-29 xenografted model and showed a strong and dose-dependent inhibitory effect on the proliferation of HT-29 cells. Mitochondrial membrane potential was reduced by TXL at the concentration of 0.5% above. For Western blot analysis, an increase in Bax expression and a decrease in Bcl-2 expression were observed in TXL-treated cells. TXL treatment increased the protein level of cleaved casepase-3 and caspase-9, and the release of cytochrome c in cytoplasm was up-regulated as well. Conclusion TXL significantly inhibits cell proliferation in the HT-29 cells and HT-29 xenografted model via the mitochondrial cell death pathway.


Background
Colorectal carcinoma increased up to four folds in the past decade and the mortality is rising [1]. Much progress has been achieved in alternative medicine [2] such as Chinese medicine.
Most methods of chemotherapy for cancer induce cancer cell apoptosis. Excessive apoptosis causes hypotrophy such as ischemic damage whereas insufficient apoptosis leads to uncontrolled cell proliferation such as cancer [3]. Chemotherapeutic agents may cause mitochondrial dysfunction leading to depolarization of the inner mitochondrial membrane potential (Δψm) [4], triggering the caspases cascade by releasing several caspase activators. Among them, cytochrome c activates caspases by forming a complex with Apaf-1 and procaspase-9, thereby triggering caspase-9 activation which subsequently cleaves the effector caspase-3 [5,6].
Tian Xian Liquid (TXL), an aqueous extraction of Chinese medicinal herbs including Radix Ginseng, Cordyceps, Radix Astragali, Radix Glycyrrhizae, Rhizoma Dioscorea, Margarita, Fructus Lycii, Ganoderma, Fructus Ligustri Lucidi, Herba Scutellariae Barbatae, has been used as an anticancer dietary supplement for more than a decade [7]. Previous experiments reported that TXL had inhibitory effects on human cervical carcinoma C-33A cells and human lung carcinoma H1299 cells [7]. The present study aims to investigate the effects of TXL on the apoptosis of HT-29 cells and tumor growth in vivo.

Preparation of TXL
Tian Xian Liquid (TXL) (Batch number: L2-171040) was provided by China-Japan Feida Union Company Ltd. and stored away from light at 4°C. TXL was diluted and incorporated into the cell culture medium RPMI 1640. Residues were removed by filtration.

Cell proliferation
Cell proliferation was assessed in vitro with 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) according to the manufacturer's protocol (Roche, USA). HT-29 cells (10000 per well) were incubated in triplicates in a 96-well plate. TXL was serially diluted with RPMI1640 and the final concentrations were 0.25, 0.5, 1, 2 and 5%. The plates were incubated with or without TXL for 24 and 48 hours. At the end of the incubation, cells were exposed to MTT (10 μL, 5 mg/ mL in phosphate-buffered saline) in culture medium for four hours at 37°C. The supernatant was removed and 150 μL DMSO (Sigma, USA) was added to dissolve the formazan crystals. The absorbance was measured at 595 nm with an ELISA plate reader (Bio-Rad, USA).

Assessment of apoptosis by determination of mitochondrial membrane potential
Mitochondrial membrane potential was assessed by 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'tetraethylbenzimidazolylcarbocyanine iodide (JC-1) according to the manufacturer's protocol (Biotium, USA). After trypsinization and centrifugation (500× g)(Eppendorf, Germany) for ten minutes at room temperature, the pellets of cell culture with or without TXL were re-suspended in RPMI 1640 medium (1 ml), stained with 5 mg/ml JC-1 for 30 minutes at 37°C in the dark, washed twice in phosphate buffered saline (PBS) and re-suspended in 0.5 ml PBS. Δψm depletion was observed under a fluorescence microscope. A green filter was used for green-fluorescent monomer at depolarized membrane potentials and a red filter for orange-fluorescent J-aggregate at hyperpolarized membrane potentials.
To measure the quantitative change of mitochondrial potential, we applied JC-1 with fluorescence plate reader. Briefly, cells (1 × 10 5 ) in 100 μl culture medium/ well were seeded in black 96-well plate (Nunc, Denmark) and treated with TXL (0.15, 0.3, 0.6, 1.25 and 2.5%). After 24 and 48 hours incubation, JC-1 (5 μg/ml) was added for the last 30 minutes of treatment. Cells were washed twice with PBS to remove unbound dye. The concentration of retained JC-1 dye was measured (490 nm excitation/600 nm emission) with a luminescence spectrometer (PerkinElmer, USA).

Western blot
The HT29 cells were incubated with increasing concentrations of TXL (0, 0.5%, 0.75%, 1%) for 48 hours. For the time-course experiment, HT29 were treated with 1% TXL for 12, 24, or 48 hours. Cellular levels of cleaved caspase-3, 9 (Cell Signaling Technology, USA) Bax/Bcl-2 cytochrome C and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, USA) were determined by Western blot. Lysates were prepared from 1 × 10 7 cells by dissolving cell pellets in 100 μl of lysis buffer. Lysates were centrifuged (Eppendorf, Germany) at 18000× g for 15 minutes and the supernatant was collected. The protein concentration was estimated with the Bio-Rad protein assay kit (Bio-Rad, USA) using bovine serum albumin as a standard. Sample proteins were resolved by 10% sodium dodecylsulfate polyacrylamide gel (Bio-Rad, USA) electrophoresis and then electrophoretically transferred to PVDF membrane (Millipore, USA) and blocked with 5% BSA (Sigma, USA). Subsequently the primary antibodies caspase-9, cleaved caspase3, Bax, Bcl-2, cytochrome C and GAPDH were added. After overnight incubation at 4°C the blots were washed, exposed to HRP-conjugated corresponding secondary antibodies for one hour and finally were visualized by ECL Advanced Solution (GE Healthcare Life Sciences, USA). Digital images were captured by Gel Doc™ gel documentation system (Bio-Rad, USA) and intensity was quantified using Quantity-One software version 4.62(Bio-Rad, USA). To evaluate the antitumor activity of TXL, we measured the tumor volume with a digital caliper six times every week (from day1 to day 6 and from day 8 to day 14) and calculated using the formula: (longest diameter) × (shortest diameter) 2 × 0.5. The body weights of all animals were recorded throughout the experiment to assess drug toxicity.

Statistical analysis
Data were presented as mean and standard deviation (SD). When one-way ANOVA showed significant differences among groups, Tukey's post hoc test was used to determine the specific pairs of groups that were statistically different. A level of P < 0.05 was considered statistically significant. Analysis was performed with the software SPSS version 16.0 (SPSS Inc, USA).

Anti-proliferative and apoptotic effects of TXL on HT-29 cells
To investigate the anti-proliferative effects of TXL on HT-29 cells, we treated the HT-29 cells with TXL in a gradient of doses (0.25-5%) and cell proliferation after two days was assessed with the MTT assay in triplicates. The results were consistent. TXL inhibited HT-29 cell proliferation in a dose-dependent manner (Figure 1). Treatment of TXL (1%) for 48 hour significantly inhibited (38.47%; P < 0.05, P = 0.011) cell proliferation.

Effects of TXL on cell nuclear morphology
Nuclear staining with DAPI was used to determine apoptosis-inducing activity of TXL in HT-29 cells. After TXL (1%) treatment, HT-29 cells underwent typical morphologic changes of apoptosis including nuclear condensation and formation of apoptosis bodies (Figure 1).

Treatment of TXL reduces the mitochondrial membrane potential
JC-1, a cationic dye, produces red fluorescent J-aggregates in mitochondria with high Δψm and green fluorescence with low Δψm. Most control cells had red J-aggregation fluorescence whereas TXL-treated cells had green fluorescence (Figure 2A). JC-1 staining was used to determine mitochondrial integrity. To quantify the change of mitochondrial potential, we applied JC-1 with fluorescence plate reader. The green to red fluorescence ratio significantly decreased at 48 hours in a dosedependent manner ( Figure 2B).

TXL triggers interaction between Bcl-2 and Bax and releases cytochrome c
Low Δψm is regulated by Bcl-2 family proteins [11]. We studied the effects of TXL on the expression of Bax and Bcl-2 which are important for mitochondrial membrane permeablization. In this study, the HT-29 cells were incubated with increasing concentrations of TXL (0, 0.5%, 0.75%, 1%) for 48 hours. For the time-course experiment, HT29 were treated with 1% TXL for 12, 24, or 48 h. Cell lysates were prepared for western blot analysis. After 48 hours, HT-29 cells treated with 1% TXL showed significant up-regulation (P = 0.003) in Bax expression ( Figure 3) while significant down-regulation (P = 0.013) in Bcl-2 expression (Figure 3). TXL (0.75%) and TXL (1%) increased the Bax/Bcl-2 ratio by 1.4 and 2.8 folds respectively in HT-29 cells. Stability of mitochondrial membrane is influenced by the interactions among Bcl-2 family proteins, thereby affecting the release of cytochrome c from mitochondria to and subsequently accumulation in the cytosol [12]. The cytosol levels of cytochrome c in HT-29 cells were examined with Western blot. In HT-29 cells treated with TXL, cytochrome c significantly increased in a dose-dependent (P = 0.0096) and time-dependent manner (P = 0.001).

TXL induces caspase-3 and caspase-9 cleavage
To confirm the induction of the mitochondrial-mediated apoptosis, we examined the activation of the intrinsic initiator caspase-9 and casapse-3 using western blot. TXL (0.75% and 1%) induced the cleavage of caspase-3 to its active form, i.e. p17 (17 kDa) which was found after 24 hours of TXL treatment ( Figure 4). As shown in Figure 4, caspases-9 in HT29 cells treated with TXL was activated, as judged by the decrease of the procaspases-9 and the increase of their cleavage products.

In vivo effects of TXL on HT-29 tumor growth
To determine the antitumor efficacy of TXL as a single agent therapy, we examined the growth of HT-29 cells in immunocompromised mice. Compared with mice orally administrated with 200 μl PBS as control group, treatment with the TXL and 5-FU significantly inhibited tumor growth ( Figure 5A). After treatment of nude mice with TXL, the tumor size was significantly (P = 0.03) decreased from day 13 to day 15. The difference in tumor size in the TXL group (P = 0.933) was not significant compared with the 5-FU group (P = 0.99).

Discussion
Most chemotherapeutic drugs induce cancer cell apoptosis whereby a cell activates its own destruction by initiating a series of cascading events including the loss of the mitochondrial transmembrane potential [6]. A rapid collapse of mitochondrial transmembrane electrical potential Δψ m is always found in chemotherapeutic agents-induced apoptosis in cancer cells [13]. The present study demonstrated that TXL-induced apoptosis was related to the collapse of the mitochondrial membrane potential Δψ m .
Our study showed the depletion of Δψm ( Figure 2) and the activation of caspase-3 of HT-29 treated with TXL. Mitochondria participate in apoptosis induction by releasing several caspase activators. Among them, cytochrome c activates caspases by forming a complex with Apaf-1 and procaspase-9, thereby triggering caspase-9 activation which subsequently cleaves the effector caspase-3 [6]. The present study found that 1% of TXL induced the cleavage of caspase-3 to its active form, namely p17 (Figure 4). The fragment, p17 (17 kDa), was accumulated after 24 hours of TXL treatment. In this study, we also observed that TXL remarkably increased the release of cytochrome c from the mitochondria to the cytosol in HT-29 cells. Levels of cytochrome c in the cytosolic fraction increased dramatically when the dosage of TXL was 0.5% or above. These results suggest a direct link between the mitochondria and the TXLinduced apoptosis.
A previous study showed that mitochondrial membrane disruption and the release of cytochrome c was controlled by Bcl-2 family protein [11]. Bcl-2 and other pro-apoptotic factors prevent mitochondrial membrane disruption while Bax promotes these events. To clarify whether Bcl-2 family was changed in TXL treated HT-29 cell to activate the release of cytochrome c, we examined the expression level of Bcl-2 and Bax with or without the TXL treatment. An increasing Bax and a decreasing Bcl-2 were observed in a time-dependent manner after exposed to 1% TXL. Our results showed that TXL induced apoptosis by increasing the Bax/Bcl-2 ratios. These observations confirmed that TXL induced apoptosis in colon cancer via the mitochondrial pathway. The above concomitant molecular events in TXLtreated HT-29 cells result in remarkable apoptosis process. Further in vivo and in vitro studies are needed to clarify the protein interactions, thereby delineating the upstream regulatory events, such as the Wnt signaling pathway which is important factor in the development of the majority of colorectal cancers [14]. TXL treated cells showed a majority of cells stained green dye due to low mitochondrial membrane potential. (B) Effect of TXL on the depolarization of HT-29 mitochondria was also measured by fluorescence plate reader using JC-1. The cells were exposed to increasing concentrations of TXL. Data represent mean and standard deviation of three individuals with asterisks denoting significant differences between controls and TXL-exposed cells (*P < 0.05, **P < 0.01). CTL: control. Protein from cytosolic fraction of HT-29 which has been treated with TXL (1%) for 12, 24 and 48 hours (C) or indicated concentration of TXL for 48 hours (D) were analyzed by Western blot with specific antibodies against cytochrome c. GAPDH antibody was used as control for equal loading. The relative expressions of proteins were quantified using Bio-Rad Quatity-One software. Results are expressed as mean and standard deviation (n = 3),*P < 0.05 **P < 0.01compared with control group. Figure 4 TXL Induces caspase-3 and caspase-9 cleavage. Protein from HT-29 which has been treated with 1%TXL for 12, 24 and 48 hours (A) and protein from HT-29 which has been treated with indicated concentration of TXL for 48 hours (B) were analyzed by Western blot with specific antibodies against caspase-9 and cleaved caspase-3. GAPDH antibody was used as control for equal loading. The relative expressions of proteins were quantified using Bio-Rad Quatity-One software. Results are expressed as means and standard deviation (n = 3),*P < 0.05 **P < 0.01 compared with control group. We studied TXL's effects on the growth of HT-29 cell lines grown in vitro and compared those results with its effect on tumor growth in vivo. We found that TXL attenuated the growth of xenografted HT-29 tumors in vivo. The injection of 5-fluorouracil (5-FU), a common choice for single-agent chemotherapy of advanced colon cancer [15,16] on nude mice significantly attenuated tumor growth ( Figure 5A). However, during the 5-FU treatment, the body weight of nude mice was significantly decreased on day 10 (P < 0.05, P = 0.038) ( Figure  5B). The components of TXL such as Radix Ginseng, Cordyceps, Radix Astragali, Fructus Lycii, Ganoderma are commonly used in China as immune-stimulating agents. Whether TXL's immunomodulation effect and protection effect on stomach and digestive system reducing toxic side effects of 5-FU will be investigated in the future.
Because 5-fluorouracil (5-FU) as common choice for single-agent chemotherapy of advanced colon cancer is also recognized for its toxicity including fatigue, diarrhea and sometimes myelosuppression, much attention has been focused on exploring complementary and alternative medicine. This study may provide a platform for evaluation the function of Chinese medicine decoctions on treatment of cancer, which has no significant side effect. To further evaluate the potential of TXL as an adjuvant agent in colon cancer chemotherapy, we are studying TXL's effect on attenuation of 5-FU-induced side effect and the synergistic anti-tumor effect of the 5-FU/TXL.

Conclusion
TXL significantly inhibits cell proliferation in the HT-29 cells and HT-29-bearing mouse model. TXL-induced apoptosis is likely achieved through the mitochondrial cell death pathway as indicated by a reduction in mitochondrial membrane potential, and the decrease of Bcl-2/Bax ratio and the release of cytochrome c followed by the activation of caspase-3 and caspase-9.