An extract from ripe fruit of Vitex agnus-castus (Vitex) has been used to treat patients with various obstetric and gynecological disorders in Europe. We have demonstrated that Vitex showed cytocidal effects on various types of cancer cell lines including a human colon carcinoma cell line, COLO 201. In this study, we extended our previous study to investigate the detailed mechanisms underlying cytocidal effects of Vi- tex on COLO 201. Furthermore, a possible clinical application of Vitex was also explored in vivo using nude mice xenografted with the cells. Treatment with Vitex induced apoptosis in COLO 201 in a time-dependent manner, accompanying with activa-tion of caspase-9 and -3, but not caspase-8. An inhibitor for c-Jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK), significantly suppressed the apoptosis induction along with caspase-3 activation. Endoplasmic reticulum (ER) stress-related genes were also upregulated by Vitex treatment. Most importantly, the in vivo efficacy of Vitex evaluated by assessing the tumor growth revealed that the administration of Vitex significantly suppressed tumor growth in COLO 201 xenografted mice. Collectively, current results suggest that apoptosis induction by Vitex in COLO 201 is mediated through the activation of JNK and caspase-9, -3 resulted from ER stress. Based on the current clinical application of Vitex, these results thus provide a new insight into the clinical use of Vitex and leave open a possibility of a new regimen as an alternative medicine approach for such devastating colon cancer treatment.
Colon cancer is one of the most common cause of cancer death worldwide [1,2]. Treatment for the recurrent and metastatic disease remains a center of clinical attention. Combinational therapy, such as 5-fluorouracil (5-FU) and leucovorin with either irinotecan or oxaliplatin has been widely used for the treatment of patients with colorectal cancer [3,4]. Recently, various types molecular targetbased drugs, such as cetuximab and bevacizumab, are being used clinically. However, there is a growing concern about the side effects of these clinically used drugs. It is a noteworthy trend that botanical therapeutics has been receiving a great attention in order to reduce chemotherapy-associated side effects. In this regard, it is interesting to note that up to 60% of cancer patients use herbal supplements during or after chemotherapy in the USA [
Vitex agnus-castus is a shrub of the Verbenaceae family and is found naturally in the Middle East and Southern Europe. Ripe fruit of V. agnus-castus has been used as a folk medicine for the treatment of various obstetric and gynecological disorders in Europe [7,8]. It has been reported that flavonoids are one of major components of an extract from dried ripe V. agnus-castus (Vitex) [
In the current study, we extended our previous study to investigate the detailed mechanisms underlying Vitexinduced apoptosis in COLO 201 cells. In order to predict a possible clinical application of Vitex, we further investtigated the effect of Vitex on the tumor growth of nude mice xenografted with COLO 201 cells.
Specific fluorogenic substrates for caspase-3 [Asp-GluVal-Asp-AFC (DEVD-AFC)], caspase-9 [Leu-Glu-HisAsp-AFC (LEHD-AFC)], caspase-8 [Ile-Glu-Thr-AspAFC (IETD-AFC)], and specific inhibitor for caspase-3, Z-DEVD-FMK, were purchased from BioVision Research Products (Mountain View, CA, USA). Boc-D-FMK, a pancaspase inhibitor, was purchased from Sigma (St. Louis, MO, USA). c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, were purchased from Calbiochem (Darmstadt, Germany).
COLO 201, a human colon carcinoma cell line [
Preparation of Vitex was carried out according to the methods described previously [
DNA preparation and agarose gel electrophoresis were carried out according to the methods previously reported [
Z-DEVD-FMK, a specific inhibitor for caspase-3, was dissolved in DMSO at a concentration of 10 mM. BocD-FMK, a pancaspase inhibitor; SP600125, a specific inhibitor for JNK; and SB203580, a specific inhibitor for p38 MAPK, were dissolved in DMSO at a concentration of 20 mM. COLO 201 cells (2 × 105 cells/ml) were cultured for 12 h prior to the addition of each of the following inhibitors, [Boc-D-FMK (final concentration: 50 μM), Z-DEVD-FMK (final concentration: 20 μM), and SP600125 or SB203580 (final concentration: 10 μM)], into culture medium, just before the addition of Vitex at a final concentration of 100 μg/ml, followed by the additional incubation for 48 h.
Activity of caspase-3, -9, or -8 was measured using the caspase fluorometric assay kit (BioVision) according to the manufacturer’s instructions. Protein amount of 50 μg/50 μl was plated on a 96-well plate, followed by the addition of 50 μl of 2 × reaction buffer containing 10 mM DTT to each sample, and then 5 µl of 1 mM caspase substrate (final concentration of 50 µM). After incubation at 37˚C for 1-h, fluorescent intensity was measured with a 400 nm excitation filter and 505 nm emission filter using a microplate reader Safire (TECAN, Männedorf, Switzerland).
Total RNA for RT-PCR analysis was extracted from Vitex-treated COLO 201 cells using an RNA extraction kit, ISOGEN (Wako Pure Chemical Industries, Osaka, Japan). Complementary DNA (cDNA) was synthesized from 1 µg of RNA using 100 pmol of random hexamers and 100 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) in a total volume of 20 µl according to the manufacture’s instruction manual. PCR was performed as described previously [
Specific pathogen-free (SPF) KSN mice (male, 5 weeks old) were purchased from Japan SLC Inc., (Shizuoka, Japan). Animal use and relevant experimental procedures were approved by the Tokyo University of Pharmacy and Life Sciences Committee on the Care and Use of Laboratory Animals. COLO 201 cells were cultured as described above, harvested, centrifuged at 1000 rpm for 3 min, washed and resuspended in sterile PBS. The total number of 5 × 106 cells in 0.2 ml was injected subcutaneously between the scapulae of each nude mouse housed under SPF condition. After transplantation, tumor size was measured using calipers and the tumor volume was estimated according to the formula: tumor volume (mm3) = L × W2/2, where L is the length and W is the width [
Experiments were independently repeated three times, and the results were shown as mean ± standard deviation (S.D.) of three assays. Student t-test as well as Scheffe’s post-hoc test was applied, and p values less than 0.05 were considered as significant.
After treatment of the cells with 100 μg/ml Vitex for an indicated time period, a typical DNA fragmentation ladder representing apoptosis induction was observed after 24 h treatment (
Addition of 10 µM SP600125, a specific inhibitor for JNK, clearly suppressed Vitex-induced apoptosis in COLO 201 cells, whereas no suppression was observed when 10 µM SB203580, a specific inhibitor for p38 MAPK, was added (Figures 2(a) & (b)). Furthermore, approximate 3-fold reduction of caspase-3 activity in Vitex-treated cells was observed in the presence of SP600125, indicating the contribution of JNK pathway to the caspase-3 activation (
After treatment with 100 μg/ml Vitex for 12 and 48 h, the expression profiles of HO-1, CHOP and GRP78 were assessed by RT-PCR. Consistent with our previous report [
gene during the treatment period (Figures 3(a) & (c)). Similarly, the expression levels of GRP78 were upregulated by the Vitex treatment, although the degree of upregulation was slightly lower than those of the other two genes (Figures 3(a) & 3(d)).
Human colon cancer xenograft was established by transplantation of COLO 201 cells into a 7-week old athymic nude mouse. Approximate required time for tumor growth to reach a mean size of 200 mm3 was two weeks. After reaching the mean size, animals received intraperitoneal injection of either 0.5 ml sterile PBS (control group) or 1 mg Vitex suspended in 0.5 ml sterile PBS to investigate anti-tumor effect of Vitex. A significant inhibition of tumor growth was observed from 26th day posttransplantation in Vitex-treated group compared to
control group (
We have been interesting in the effects of naturally derived flavonoids on cancer cell growth, particularly malignant cells in the gastrointestinal tract. In this regard, we have previously demonstrated that a crosstalk between intrinsic and extrinsic pathway via Bid activation as a result of oxidative stress plays a critical role in Vitexinduced apoptosis in KATO-III cell line, a human gastric signet ring cell carcinoma [
In this study, apoptosis induction was reconfirmed in Vitex-treated COLO 201 cells, accompanying with activation of caspase-9 and -3, but not caspase-8 (Figures 1(a) & 1(d)). Furthermore, both a pancaspase inhibitor and a specific caspase-3 inhibitor clearly suppressed the apoptosis induction (Figures 1(b) & 1(c)), suggesting the involvement of caspase pathways in the apoptosis induction. We also demonstrated that the addition of a specific inhibitor for JNK not only clearly inhibited the activation of caspase-3 but also apoptosis induced by Vitex treatment, whereas it was not observed when a specific inhibitor for p38 MAPK was added (
important component of Vitex [
It is well-known that JNK pathway is involved in the regulation of caspase-3 via activation of caspase-8 [24, 25]. However, the activation of caspase-8 was not observed in the Vitex-treated COLO 201, while the activetion was observed in Vitex-treated KATO-III [
We further demonstrated that the expression of HO-1
gene was upregulated substantially by Vitex treatment (Figures 3(a) & 3(b)), similar to our previous report [
Most importantly, our in vivo experimental data revealed that the administration of Vitex significantly suppressed tumor growth in COLO 201 xenograft mice (
Our results suggested that the activation of JNK and caspase-9 and -3 resulted from ER stress contributed to the apoptosis induction in Vitex-treated COLO 201 cells. Furthermore, we recently reported for the first time that 5-FU in combination with Vitex achieved an enhanced cytocidal effect on the cells, but lesser cytotoxic effect on human peripheral blood mononuclear cells [
We thank Makoto Origuchi, Hirokazu Ishikawa and Tetsuya Kimata for their technical assistance. We are also grateful to Dr. Yoshinori Nozawa for excellent advises. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology and by the Promotion and Mutual Aid Corporation for Private School of Japan.