Re:Re:Re:Mechanism of taurine-induced apoptosis in human colon cancer cells

[문형철]

beyond reason

타우린은 angiogenesis 자극효과가 있어서 암에 부정적인 보충제????

논문상에는 apoptosis를 일으켜 항암효과

Mechanism of taurine-induced apoptosis in human colon cancer cells 

Xiali Zhang, Shuo Tu, Yibing Wang, Baohua Xu, Fusheng Wan 

Author Notes

Acta Biochimica et Biophysica Sinica, Volume 46, Issue 4, April 2014, Pages 261–272, https://doi.org/10.1093/abbs/gmu004

Published:

 

07 March 2014

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Abstract

Taurine (Tau) has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anti-cancer effect is not well understood. PUMA (p53-upregulated modulator of apoptosis) plays an important role in the process of apoptosis induction in a variety of human tumor cells in both p53-dependent and -independent manners. However, whether PUMA is involved in the process of Tau-induced apoptosis in cancer cells has not been well studied. In the present study, we treated human colorectal cancer cells HT-29 (mutant p53) and LoVo (wild-type p53) with different concentrations of Tau, which led to the repression of cell proliferation and induction of apoptosis in both cell lines. Meanwhile, we also observed the increased expression‎ of PUMA and high Bax/Bcl-2 ratios. To determine the role of PUMA in Tau-induced apoptosis, we used small interfering RNA interference to suppress PUMA expression‎. As a result, apoptosis was decreased in response to Tau treatment. All these results indicated that PUMA plays a critical role in Tau-induced apoptosis pathway in human colorectal cancer cells. Demonstration of the molecular mechanism involved in the anti-tumor effect of Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.

taurine, human colon cancer cell, PUMA

Topic:

 

• apoptosis
• cell proliferation
• colorectal cancer
• cell lines
• bcl2 gene
• ht29 cells
• taurine
• neoplasms
• mapt gene
• tumor cells, malignant

Issue Section:

 Original Articles

Introduction

Taurine (Tau; 2-aminoethanesulfonic acid) was first reported as an essential nutrient for cat in 1975 [1]. A large number of studies and clinical applications demonstrated that Tau has many extensive physiological effects, so it was identified as an endogenous anti-injury material. Recently, Tau has been used as an antipyretic and anti-inflammatory agent, to treat liver and gallbladder disease [2], cardiovascular disease [3], diabetes [4,5], cataract [6,7], and so on. But its effect on tumors has not been previously studied. Tau could protect cells from oxidant-induced injuries by scavenging strong oxidant and cytotoxic agents [8], therefore, Tau as an effective antioxidant, may hinder the increase of reactive oxygen species (ROS) in tumors, leading to a delay of the development of cancer [9]. In addition, Tau could play a role in the process of anti-tumors by down-regulating MMP-2, up-regulating N-acetylgalactosaminyltransferase, and meanwhile inhibiting potential invasion and metastasis induced by ionizing radiation [10].

Apoptosis, also known as programmed cell death, is an autonomous cell death based on a genetic program. Bcl-2 family members exert their effects through mitochondrial apoptosis pathway which plays a pivotal role in the apoptotic pathways. Until now, the members of Bcl-2 family can be divided into anti-apoptotic (including Bcl-2, Bcl-xL) and pro-apoptotic subsets (including Bax, Bak, Bid, Bad, Bim, and so on) according to their structures and functions. The Bcl-2 family members share homology in the 1–4 conserved regions which are designated as the Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4, respectively. Among them, the BH3 domain with the amphipathic α-helical structure plays an important role in apoptosis by mediating the interaction among the Bcl-2 family members. Once cells are exposed to apoptotic stimuli, the BH3-only protein and other regulators are translocated to mitochondria, where they promote the release of pro-apoptotic factors (including cytochrome C, Smac/DIABLO, AIF, and endonuclease G) through changing the mitochondrial permeability.

PUMA (p53-upregulated modulator of apoptosis), a potent pro-apoptotic protein, is a member of the BH3-only subgroup of Bcl-2 family proteins. Once exposed to a variety of apoptotic stimuli, the expression‎ of intracellular PUMA would be increased. BH3 domain of PUMA directly interacted with anti-apoptotic Bcl-2 family members, inhibiting the interaction with the pro-apoptotic molecules. Thus, PUMA was suggested to be the most potential regulator that triggers apoptosis [11,12]. PUMA induced cell apoptosis by the following three mechanisms: (i) directly binding and oligomerizing Bax/Bak, leading to conformational change and mitochondrial translocation, finally changing the original ‘membrane channel gene’; (ii) indirectly activating Bax and/or Bak by relieving them from the inhibition effect of anti-apoptotic Bcl-2/Bcl-xL; (iii) binding to Bcl-xL, which releases p53 to directly activate Bax. These pathways increase the permeability of the mitochondrial membrane and the release of pro-apoptotic factors, and finally initiate the cell apoptosis. PUMA is an essential apoptotic mediator of p53-dependent and -independent pathways [13] and was reported to have a potential binding site for p53 in its promoter region. In p53-dependent apoptosis pathway, within hours of apoptosis stimuli, p53 was recruited to p53-responsive element, and furthermore the transcription and translation of PUMA were induced [14,15]. Meanwhile, PUMA could be induced by other regulators in a p53-independent manner, such as glucocorticoids [16], transcription factor E2F1 [17], and c-Jun kinase [18]. Several studies have shown that PUMA plays important roles in tumors [19], cerebral vascular and myocardial hypoxia reoxygenation injury [20,21], and cell apoptosis of multiple cell lines. Therefore, PUMA might be an efficient target for cancer therapy.

In the present study, we treated human colorectal cancer cells HT-29 and LoVo with Tau, which led to repression of cell proliferation and induction of apoptosis in both cell lines. The results indicated that PUMA plays a critical role in Tau-induced apoptosis pathway in human colorectal cancer cells and Tau may be useful in the therapeutic target selection for p53-deficient colorectal cancer.

Materials and Methods

Reagents and antibodies

Tau was purchased from Sigma (St Louis, USA). RPMI-1640 medium, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum were purchased from Gibco (Gaithersburg, USA). cDNA reverse transcription kit was purchased from Thermo Scientific (Waltham, USA). Anti-GAPDH polyclonal antibody (TA-08), anti-Bcl-2 monoclonal antibody (sc-783), and anti-Bax monoclonal antibody (sc-526) were purchased from Santa Cruz (Santa Cruz, USA). Anti-PUMA polyclonal antibody (#Q9BXH1-4976) was purchased from Cell Signaling Technology (Beverly, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZDR-5307) and goat anti-rabbit IgG (ZDR-5306) were purchased from Beijing ZSGB-Bio Company (Beijing, China).

Cell lines and animals

Human colon cancer cells LoVo (p53+/+), human kidney epithelial cells 293T, and human colon cancer cells HT-29 (p53−/−) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM and RPMI-1640 medium containing 10% fetal bovine serum, respectively. All cell lines were maintained at 37°C with 5% CO₂. Female-specific pathogen-free nude mice (BALB/C) (certificate No. HNASLKJ20121369), 4 weeks old, were purchased from Hunan Slack King of Laboratory Animal (Changsha, China) and kept in a laminar flow cabinet (Su net Aetna, Suzhou, China) in the Department of Animal Science, Nanchang University, at constant temperature, constant humidity, and sterile environment.

Drug treatment

All cell lines were treated with concentration gradient of Tau (10–160 mM, dissolved in medium without fetal bovine serum) for different time periods. The cells treated with 4 μg/ml of cisplatin (DDP) were used as positive control.

Cell proliferation analysis

Cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Sigma (St Louis, USA). Briefly, 5000–8000 cells per well were seeded into a 96-well culture plate in quadruplicate for each group. After an overnight incubation, the cells were treated with various concentrations of Tau (10–160 mM) and incubated for 24, 48, and 72 h, respectively. MTT reagent [20] was added to each well at 5 mg/ml and the mixture was incubated for another 4 h. The formazan crystals formed by the viable cells were then dissolved in 150 μl dimethyl sulfoxide and the absorbance values were measured at 490 nm. The inhibition rate was calculated as follows: 

Inhibitionrate(%)=(1−absorbanceoftheexperimentgroup/absorbanceofthecontrolgroup)×100%$$\begin{array}{rl}& \text{Inhibition}\text{rate}(\mathrm{\%})=(1-\text{absorbance}\text{of}\text{the}\text{experiment}\\ & \text{group}/\text{absorbance}\text{of}\text{the}\text{control}\text{group})\times 100\mathrm{\%}\end{array}$$

Flow cytometry analysis

Apoptosis was analyzed by detecting phosphatidylserine externalization with flow cytometry. Two-color analysis with fluorescein isothiocyanate (FITC)-labeled Annexin V/PI double staining was used (the Annexin V-FITC/PI kit was a product of Nanjing Key-GEN BioTECH Co., Nanjing, China). After being treated with Tau for 48 h, 1–5 × 105 cells were collected and suspended in 500 µl binding buffer and incubated with FITC-labeled Annexin V (BioLegend, San Diego, USA) and propidium iodide (PI) for 15 min in the dark at room temperature. Apoptosis were then analyzed by fluorescence-activated cell sorting (Becton Dickinson, Franklin Lakes, USA).

Hoechst 33342 staining

The cell suspension was seeded in 12-well plate at 1 × 105 cells per well. In the next day, the cells were treated with 80 mM of Tau for 48 h. A total of 500 µl of Hoechst 33342 (Beyotime, Haimen, China) was added to each well and incubated for 20–30 min at 37°C in the dark. Finally, the unbound dye was removed and the morphology of apoptotic cells was observed using a fluorescence microscope (Olympus, Tokyo, Japan).

Reverse transcription-polymerase chain reaction analysis

Total RNA was routinely extracted using Trizol (Invitrogen, Carlsbad, USA). RNA concentration and purity were measured using an Eppendorf nucleic acid quantitative determination meter. Then, RNA integrity was observed after gel electrophoresis. GAPDH was used as an internal control. PUMA, Bax, and Bcl-2 expression‎ levels were detected by reverse transcription-polymerase chain reaction (RT-PCR). The primers of PUMA, Bax, Bcl-2, and GAPDH are listed in Table 1. PCR amplification products were analyzed in 1.5% agarose gel electrophoresis. Gel images were taken for strap gray scale analysis. Optical density (OD) values of the target genes and the corresponding GAPDH products in each group were detected. The ratio between the target gene and GAPDH was statistically analyzed.

Table 1.

The primer sequences for RT-PCR

Gene	Primer sequence (F/R)	Length of product (bp)
PUMA	5′-CCTCAGCCCTCGCTCTCGC-3′/ 5′-CCGATGCTGAGTCCATCAGC-3′	559
Bcl-2	5′-GTGGAGGAGCTCTTCAGGGA-3′/ 5′-AGGCACCCAGGGTGATGCAA-3′	304
Bax	5′-TTGCTTCAGGGTTTCATCCA-3′/ 5′-CAGCCTTGAGCACCAGTTTG-3′	298
GAPDH	5′-GGCTCTCCAGAACATCAT-3′/ 5′-CACCTGGTGCTCAGTGTA-3′	241
GAPDH	5′-TCACCATCTTCCAGGAGCGAG-3′/ 5′-TGTCGCTGTTGAAGTCAGAG-3′	648

F, forward primer; R, reverse primer.

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Western blot analysis

Total protein was extracted from the treated cells in each group. After BCA protein assay reagent (Pierce, Rockford, USA) quantitation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the protein sample was electrotransferred to a polyvinylidene difluoride membrane (Millipore, Massachusetts, USA). The membranes were incubated with primary antibodies and then with secondary antibodies. Finally, the protein signals were detected by the enhanced chemiluminescence detection system (Pierce). The gray scale of protein bands were scanned using Image-Pro Plus 6.0. The expression‎ relative to GAPDH was analyzed.

Spectrophotometry analysis

The caspase-9 and caspase-3 activity of the colon cancer cells was detected using the spectrophotometric method. The analysis was performed according to the instruction of kit (Beyotime).

Transient transfection analysis

PUMA-specific siRNA (5′-GGGUCCUGUACAAUCUCAUTT-3′, 5′-AUGAGAUUGUACAGGACCCTT-3′) was designed and synthesized by Shanghai GenePharma Company (Shanghai, China) according to the sequence of human PUMA cDNA. After PUMA siRNA (si-PUMA) was transfected into LoVo cells by Lipofectamine 2000 (Invitrogen), the effect of Tau on LoVo cell apoptosis and the changes of relative expression‎ were detected.

In vivo Tau studies in subcutaneous tumor models

Four-week-old female nude mice (four in each group) were subcutaneously injected with 0.2 ml of LoVo and HT-29 cells (5.0 × 107 cells/ml) in the right and left flanks. Nude mice were treated every 48 h for seven times when the diameter of tumor tissue was ∼2–3 mm. We obtained five groups: the control (intraperitoneal injection 0.1 ml), the Tau low-dose group (intraperitoneal injection 0.1 ml Tau, 50 mg/kg), the Tau middle-dose group (intraperitoneal injection 0.1 ml Tau, 100 mg/kg), the Tau high-dose group (intraperitoneal injection 0.1 ml Tau, 200 mg/kg), and the positive control group (intraperitoneal injection 0.1 ml DDP, 5 mg/kg). Tumor volumes were measured every 5 days. After 25 days, mice were killed and tumor weights were measured.

Statistical analysis

The results were expressed as the mean ± SD. Data were analyzed by the one-way analysis of variance test by SPSS 17.0 software. P < 0.05 was considered as statistically significant.

Results

Tau inhibits cell proliferation in LoVo and HT-29 cells, but not in 293T cells

To investigate the potential cell growth inhibition by Tau in colon cancer, we first examined the effect of Tau on cell proliferation in LoVo and HT-29 cells. Tau significantly inhibited cell growth in both cancer cell lines in concentration-dependent and time-dependent manners (Fig. 1A,B). At 24, and 72 h, the IC₅₀ of Tau for LoVo and HT-29 cells were 627.88, 297.81, 161.31, 253.69, 62.57, and 45.66 mM, respectively. By calculating the IC₅₀ values, we showed that the inhibitory effect of Tau was more evident on p53−/− cancer cells than that on p53+/+ cancer cells. To eliminate Tau toxicity on cell growth, kidney epithelial cells 293T were used as the control with the same dose and treated time. The results showed that Tau, even at the highest dose of 160 mM, had no effect on the cellular viability of 293T cells (Fig. 1C).

Figure 1.

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Tau inhibits the cancer cell growth in human colorectal cancer cells in vitro, but not in 293T cells Cells were treated with various concentrations of Tau (10–160 mM) for the indicated time (24, 48, and 72 h), followed by the assessment of the inhibition rate by MTT assay. The data were expressed as the mean ± SD of three independent experiments. **P < 0.01 vs. Tau 0 mM group. (A) LoVo cells. (B) HT-29 cells. (C) OD values of 293T cells.

Tau induces apoptosis in LoVo and HT-29 cells

FITC Annexin-V/PI double staining was performed to examine the apoptotic rate of both cell lines treated with Tau. After Tau treatment for 48 h, the rate of inhibition in both cell types were significantly increased in a concentration-dependent manner, and the pro-apoptotic effect of Tau was more significant in p53−/− cancer cells than that in p53+/+ cancer cells (Fig. 2A–D). To detect the morphology in apoptotic cells, Hoechst 33342 fluorescent staining was performed after Tau (80 mM) treatment. Untreated cells stained by Hoechst 33342 had round and light-blue nucleus with high cell density. On the contrary, the Tau-treated cells possessed lower cell density and more apoptotic cells with densely stained. Typical feature of apoptosis such as apoptotic body was observed (Fig. 2E).

Figure 2.

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Tau induces cell apoptosis in LoVo and HT-29 cells LoVo and HT-29 cells were treated with Tau (40, 80, and 160 mM) for 48 h, and apoptotic cells were analyzed by flow cytometry. (A) The statistical analysis of the apoptosis rate of LoVo cells. (B) The statistical analysis of the apoptosis rate of HT-29 cells. (C) Apoptosis rate of different groups of LoVo cells. (a) Control group. (b) Tau 40 mM. (c) Tau 80 mM. (d) Tau 160 mM. (D) Apoptosis rate of different groups of HT-29 cells. (a) Control group. (b) Tau 40 mM. (c) Tau 80 mM. (d) Tau 160 mM. Data from triplicate experiments were collected on a histogram (n = 3). **P < 0.01 vs. control group. (E) The change of morphology in human colon cancer cells by Hoechst33342 (Magnification, ×200). (a) Control group of LoVo cells. (b) Tau 80 mM group of LoVo cells. (c) Control group of HT-29 cells. (d) Tau 80 mM group of HT-29 cells.

Tau induces the expression‎ of apoptosis-associated gene

The results of RT-PCR assay showed that expression‎ of PUMA gene was significantly up-regulated in both cells with increasing concentration of Tau. For other apoptosis-related genes, the expression‎ of Bax was up-regulated while Bcl-2 was down-regulated, and the Bax/Bcl-2 ratios were increased (Fig. 3).

Figure 3.

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RT-PCR analysis of PUMA, Bax, and Bcl-2 in human colon cancer cells (A) mRNA expression‎ in LoVo cells. (a) RT-PCR analysis of PUMA, Bax, and Bcl-2 in LoVo cells treated by Tau. (b–d) Relative levels of PUMA, Bax, and Bcl-2 mRNA expression‎ in LoVo cells, normalized to GAPDH expression‎. Relative level of mRNA expression‎ in the control group was preset to 1. (e) The changes of Bax/Bcl-2 ratios in LoVo cells. The ratio in the control group was preset to 1. (B) mRNA expression‎ in HT-29 cells. (a) RT-PCR analysis of PUMA, Bax, and Bcl-2 in HT-29 cells treated by Tau. (b–d) Relative levels of PUMA, Bax, and Bcl-2 expression‎ in HT-29 cells, normalized to GAPDH expression‎. Relative level of mRNA expression‎ in the control group was preset to 1. (e) The changes of Bax/Bcl-2 ratios in HT-29 cells. The ratio in the control group was preset to 1. Values are expressed as the mean ± SD (n = 3). *P < 0.05 vs. control group.

Western blot analysis was performed to examine the expression‎ changes of apoptosis molecules at the protein levels. Both cell lines treated with Tau exhibited an increase of PUMA and Bax and a decrease of the Bcl-2. So, the Bax/Bcl-2 ratios were increased (Fig. 4).

Figure 4.

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Western blot analysis of PUMA, Bax, and Bcl-2 in human colon cancer cells (A) Protein expression‎ of LoVo cells. (a) Western blot analysis of PUMA, Bax, and Bcl-2 in LoVo cells treated by Tau. (b–d) Relative expression‎ levels of PUMA, Bax, and Bcl-2 proteins in LoVo cells, normalized to GAPDH expression‎. Relative expression‎ levels of proteins in the control group were preset to 1. (e) The changes of Bax/Bcl-2 ratios in LoVo cells. The ratio in the control group was preset to 1. (B) Protein expression‎ of HT-29 cells. (a) Western blot analysis of PUMA, Bax, and Bcl-2 in HT-29 cells treated by Tau. The PUMA antibody also cross-reacts with an 18 kDa band of unknown origin. (b–d) Relative expression‎ levels of PUMA, Bax, and Bcl-2 proteins in HT-29 cells, normalized to GAPDH protein. Relative expression‎ levels of proteins in the control group were preset to 1. (e) The changes of Bax/Bcl-2 ratios in HT-29 cells. The ratios in the control group were preset to 1. Values are expressed as the mean ± SD (n = 3). *P < 0.05 vs. control group.

Caspase-9 and caspase-3 activity in the control group was set to 1. Spectrophotometry analysis showed that caspase-9 and caspase-3 activities in LoVo and HT-29 cells were significantly increased after Tau treatment (Fig. 5).

Figure 5.

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Tau activates caspases activity in human colon cells (A) LoVo cells were treated with Tau (40, 80, and 160 mM) for 48 h, and caspase-9 activity was analyzed by spectrophotometric analysis. (B) Caspase-3 activity in LoVo cells. (C) Caspase-9 activity in HT-29 cells. (D) Caspase-3 activity in HT-29 cells. Date are expressed as the mean ± SD (n = 3). *P < 0.05 vs. control group.

Effect of Tau on LoVo cells after small interfering RNA-specific silencing of PUMA expression‎

After small interfering RNA (siRNA)-specific interference of PUMA expression‎, LoVo cells were treated with 80 mM of Tau for 48 h and then used for analysis of apoptotic rate. The results showed that the apoptotic rate was significantly lower at early/late stage in the si-PUMA group than that in the control group, whereas the apoptotic rate was increased about 2% at late apoptotic rate in the si-PUMA + Tau group, when compared with that in the control group, which might be due to the toxicity of Lipofectamine 2000. The early/late apoptotic rate was significantly higher in the si-PUMA + Tau group than that in the si-PUMA group (Fig. 6A,B). The difference of the early apoptotic rate of the si-PUMA + Tau group was significant, when compared with that of the Tau 80 mM group (Fig. 2C). After specific interference of PUMA expression‎ in LoVo cells, the expression‎ of PUMA and Bax proteins was down-regulated while the Bcl-2 protein expression‎ was up-regulated in the si-PUMA group. After silencing of PUMA, the LoVo cells were continuously treated with 80 mM of Tau. The results showed that, compared with the si-PUMA group, the expression‎ of PUMA and Bax proteins was up-regulated while the Bcl-2 protein expression‎ was down-regulated in the si-PUMA + Tau group (Fig. 6C,D).

Figure 6.

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Effect of Tau on LoVo cells after siRNA-specific silencing of PUMA expression‎ (A) Data from triplicate experiments were collected on a histogram. (B) Apoptotic cells were analyzed by flow cytometry. (a) Control group. (b) Negative group (NC). (c) si-PUMA group. (d) si-PUMA + Tau group. The percentage of apoptotic cells was calculated with WinMDI software. **P < 0.01 vs. control group; ##P < 0.01 vs. si-PUMA group. (C) Relative expression‎ levels of PUMA, Bax, and Bcl-2 proteins, normalized to GAPDH protein. (D) Results from western blot analysis. Values are expressed as the mean ± SD (n = 3), *P < 0.05 vs. control group; #P < 0.05 vs. si-PUMA group.

Effect of Tau on the growth of human colon cancer cells in nude mice

To examine the effect of Tau on human colon cell growth, we performed a simulation of Tau therapy experiments in nude mice. Nude mice were subcutaneously inoculated with human colon LoVo and HT-29 cells. When the diameter of tumor tissue was ∼2–3 mm, Tau and DDP were used to treat nude mice xenografts. We measured tumor volumes every 5 days and made a growth curve (Fig. 7A,B). After 25 days, mice were killed, tumors were excised (Fig. 7C), and the tumor weights were measured. The results showed that there were significant differences between the Tau 50 mg/kg, Tau 100 mg/kg, Tau 200 mg/kg, respectively, and the DDP 5 mg/kg groups of LoVo and HT-29 cells with regard to the average tumor volume and the average tumor weights as shown in Table 2. The inhibition rate was increased with the increase of Tau concentration, and the inhibitory effect of Tau was more evident in HT-29 cells-nude mice xenografts than in LoVo cells (Fig. 7D).

Table 2.

The average tumor volume, weight, and tumor inhibition rate of human colon cells xenografts after 25 days of treatment (⁠x¯$\overline{x}$ ± s, n = 4)

Groups	Average tumor volume (cm3)		Average tumor weight (g)		Tumor inhibition rate (%)
	LoVo	HT-29	LoVo	HT-29	LoVo	HT-29
Control	2.91 ± 0.21	2.93 ± 0.24	2.29 ± 0.18	2.30 ± 0.20	–	–
Tau 50 mg/kg	2.58 ± 0.23*	2.50 ± 0.20*	2.17 ± 0.20*	2.08 ± 0.18*	5.24 ± 0.38	9.57 ± 0.78a
Tau 100 mg/kg	2.03 ± 0.15*	1.97 ± 0.14*	1.64 ± 0.14*	1.44 ± 0.11*	28.38 ± 2.04	37.39 ± 3.26b
Tau 200 mg/kg	1.70 ± 0.12*	1.49 ± 0.10*	1.39 ± 0.10*	1.09 ± 0.64*	39.30 ± 2.97	52.61 ± 4.83c
DDP 5 mg/kg	1.33 ± 0.11*	1.30 ± 0.09*	0.56 ± 0.04*	0.54 ± 0.04*	75.55 ± 5.44	76.52 ± 5.71

*P < 0.05 vs. control group.

aP < 0.05 vs. Tau 50 mg/kg group of LoVo cells.

bP < 0.05 vs. Tau 100 mg/kg group of LoVo cells.

cP < 0.05 vs. Tau 200 mg/kg group of LoVo cells.

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Figure 7.

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Effect of Tau on the growth of human colon cancer cells-nude mice xenografts (A) The growth curve of tumor in nude mice bearing LoVo cells. (B) The growth curve of tumor in nude mice bearing HT-29 cells. (C) Tumors in nude mice bearing human colon cancer HT-29-cell xenografts. (D) The tumor inhibition rates induced by Tau and DDP in human colon cancer cells-nude mice xenografts, aP < 0.05 vs. Tau 200 mg/kg group of LoVo cells. bP < 0.05 vs. Tau 100 mg/kg group of LoVo cells. cP < 0.05 vs. Tau 50 mg/kg group of LoVo cells.

Discussion

It is currently thought that Tau exerts an anti-tumor effect mainly through four molecular mechanisms: (i) Tau has an efficacy-enhancing and toxicity-reducing effect on chemotherapy drugs. It has been reported that Tau has an efficacy-enhancing and toxicity-reducing effect on cyclophosphamide [22] and improve the efficacy of cyclophosphamide in the treatment of leukemia [23]. Tau can improve the anti-tumor efficacy of doxorubicin without increasing the side effects [24]. (ii) Tau inhibits cancer growth by improving the antioxidant capacity in vivo. In B16F10 melanoma cells, Tau can increase the activities of superoxide dismutase (SOD), glutathione peroxidase, and catalase, reduce the concentration of ROS in a dose-dependent manner, and further inhibit the growth of cancer cells [25]. In addition, Tau can increase the SOD activity in mouse heart and brain tissues, leading to the decrease in malondialdehyde content. (iii) Tau plays a role in immune rejection of cancer by enhancing the immune system in vivo. It has been indicated that Tau improves the proliferation rate of blood lymphocyte and the proliferation index of spleen lymphocyte in S₁₈₀ cancer-bearing mice, and to a certain extent increases the ratios of organ to body of spleen and thymus [26]. (iv) Tau inhibits cancer growth by inducing apoptosis in cancer cells. It has been reported that Tau Schiff base copper complex potently inhibits the activity of purified 20S and 26S proteasomes in human breast cancer MDA-MB-231 and leukemia Jurkat T-cells, which results in the accumulation of ubiquitinated protein and the proteasome target proteins p27 and Bax, followed by the induction of apoptosis [27].

PUMA is of great importance in the drug-induced cancer cell apoptosis. A variety of chemotherapy drugs can activate the PUMA expression‎, further inducing apoptosis. It has been reported that DDP-induced PUMA expression‎ is time- and dose-dependent and its effect on inducing apoptosis in rectal cancer cells is suppressed after antisense lipofection [28]. Now, traditional Chinese medicines have gained increasing attention. A variety of ingredients of traditional Chinese medicines have been shown to have anti-tumor effects, and their pro-apoptotic role is closely related to the PUMA expression‎. Previous studies demonstrated that the PUMA expression‎ was increased during the allicin induction of apoptosis in colon cancer LoVo cells. When PUMA expression‎ was inhibited, the effect of allicin on inducing apoptosis was significantly weakened. The same results were observed by Wang et al. [29], who treated the colon cancer LoVo cells with green tea polyphenol. In the prostate cancer LNCaP cells treated with wogonin, the intracellular PUMA protein expression‎ was increased [30].

In the present study, the anti-tumor effect of Tau was investigated at the perspective of apoptosis. The results showed that Tau inhibited the cell proliferation and induced cell apoptosis in human colon cancer cells, and Tau could inhibit the tumor growth in human colon cancer cells-nude mice xenografts. We further explored the molecular mechanisms of Tau-induced apoptosis in cancer cells. The results showed that Tau probably up-regulated the PUMA expression‎, up-regulated the Bax expression‎, and down-regulated the Bcl-2 expression‎, finally leading to the increased activity of caspase-3/9. These findings provide a new molecular basis for the anti-tumor effect of Tau. More importantly, our results demonstrated that Tau did not reduce the activity of human embryonic kidney 293T cells. This observation has significant implications because an ideal chemopreventive agent should be able to eliminate cancer cells while exerting nearly no toxic effect on normal cells. In addition, Tau not only induced cell apoptosis but also increased the PUMA expression‎ in the human colon cancer LoVo and HT-29 cells. These results suggested that the effect of Tau on inducing the PUMA expression‎ is unrelated to the status of p53. Interestingly, Tau had more significant effects on inhibiting the proliferation and inducing the apoptosis in p53−/− cancer cells than those in p53+/+ cancer cells, and the same result was obtained in the human colon cancer cells-nude mice xenografts. The detailed molecular mechanism remains to be elucidated.

Through RNAi, we observed that the apoptotic rate of LoVo cells was decreased in the si-PUMA transfection group. Further treatment with 80 mM of Tau resulted in obvious increase of the apoptotic rate in LoVo cells, but the increasing effect was lower than that of 80 mM of Tau treated alone. When the PUMA protein was significantly down-regulated in LoVo cells of the si-PUMA group, the corresponding expression‎ level of Bax protein was substantially down-regulated and the expression‎ level of Bcl-2 protein was significantly increased compared with the control group. Further treatment with 80 mM of Tau resulted in slight up-regulation of the PUMA and Bax protein levels and slight down-regulation of the Bcl-2 protein level in the si-PUMA + Tau group compared with the si-PUMA group. Although Tau still up-regulated the PUMA expression‎ in cancer cells after exogenous interference by si-PUMA, its up-regulation (28% relative to the si-PUMA group) was obviously lower than that of 80 mM Tau treated alone (95.3% relative to the si-PUMA group). In addition, the important pro-apoptotic effect of Tau was significantly reduced. Taken together, these results indicated that PUMA acts as a key molecule in Tau-induced cancer cell apoptosis, and Tau prompts the transcription and translation of the PUMA gene. However, further study is needed to examine whether Tau promotes the PUMA gene transcription via the activation of PUMA promoter or stimulation of PUMA expression‎ through other pathways.

Hence, the apoptotic rate of LoVo cells under normal circumstances can be substantially decreased by transfection with chemically synthesized si-PUMA. The primary mechanism might be the fact that si-PUMA inhibits the transcription of PUMA and effectively interferes with its pro-apoptotic function, leading to the down-regulation of Bax protein and up-regulation of Bcl-2 protein, eventually preventing cell apoptosis in LoVo cells. These data further demonstrated that PUMA is a key molecule that mediates the Tau-induced apoptosis in LoVo cells, and si-PUMA treatment is resistant to the effect of Tau on inducing cancer cell apoptosis. Therefore, people can take advantage of the foreign gene transduction of Ad-PUMA or stimulation by chemotherapy drugs to appropriately increase the PUMA expression‎, further improving the anti-tumor effect of Tau.

In this study, the anti-tumor effect of Tau on human colon cancer cells was examined. The results showed that Tau significantly inhibited the cell proliferation of colon cancer cells and promotes the occurrence of apoptosis. Regarding the molecular mechanisms, Tau may induce apoptosis in cancer cells through two pathways: (i) stimulation of the increased PUMA expression‎; and (ii) up-regulation of the pro-apoptotic Bax gene expression‎ and down-regulation of the anti-apoptotic Bcl-2 gene expression‎, finally leading to the increased activity of caspase-3/9. RNAi results demonstrated that PUMA is a key molecule that mediates the Tau-induced cancer cell apoptosis. In addition, Tau has an obvious effect on inhibiting the p53 mutant cancer cells, suggesting that Tau can up-regulate the PUMA gene expression‎ in the abovementioned two groups of cells, and up-regulation of PUMA gene expression‎ by Tau is not dependent on the status of p53.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (30060029) and Natural Science Foundation of Jiangxi Province (2010JXY0237).

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