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Cell Biology International (2006) 30, 940946 (Printed in Great Britain)
A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release
Zhonghai Liab* and Jingze Wanga*
aState Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, 25 Beisihuan Xi Road, 100080 Beijing, China
bChinese Human Genome Research Center, 250 Bibo Road, 201203 Shanghai, China
FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Δψ
Keywords: FSK88, Apoptosis, Bcl-2, Cytochrome c, Caspases.
*Corresponding authors. Present address: 12 Binnenhaven, 6709 PD, Wageningen, The Netherlands. Tel.: +31 317 482 324; fax: +31 317 484 821 (Zhonghai Li). 25 Beisihuan Xi Road, 100080 Beijing, China. Tel.: +86 10 6255 1668; fax: +86 10 6256 5689 (Jingze Wang).
Gastric cancer is the second leading cause of cancer death in the world and particularly prevalent in certain countries including China (Bamias and Pavlidis, 1998; Hohenberger and Gretschel, 2003; Lu et al., 2005). However, its pathogenesis is not completely understood and there are few effective therapies in gastric cancer prevention and treatment (Hampton and Orrenius, 1997). The prognosis of gastric cancer is poor, with a five-year survival of 15%–20% (Thompson et al., 1993). Cancer of the stomach is a disease for which treatment and attitudes vary in different regions of the world. That an organ cancer should show varying causative factors in different parts of the world is not unusual; however, with gastric cancer, it is not only the incidence of the disease, but also the approach of early diagnosis and treatment that varies greatly between the western and eastern hemispheres (Hohenberger and Gretschel, 2003). Currently, inducing cancer cells into apoptosis is one of the important therapeutic intervention approaches in cancer (Ferreira et al., 2002; Hengartner, 2000; Kasibhatlal and Tseng, 2003; Lowe and Lin, 2000; Tamm et al., 2001a,b), therefore, it is crucial to reveal the molecular mechanism of apoptosis in gastric cancer cells. In this study, BGC823 cells, a human gastric cancer cell line, was exploited to investigate the effects of FSK88 on the human gastric cancer cells and the underlying molecular mechanisms.
It has been reported that forskolin, a diterpene from the roots of the herb Coleus forskohlii, is capable of preventing tumor colonization and metastasis (Agarwal and Parks, 1983) and inhibiting growth and inducing apoptosis of myeloid and lymphoid cells (Gutzkow et al., 2002; Moon and Lerner, 2003; Taetle and Li-en, 1984). Recently, it has been found that forskolin, as a potent PP2A activator (Feschenko et al., 2002; Neviani et al., 2005), induced marked apoptosis, reduced proliferation, impaired colony formation, inhibited tuomorigenesis, and restored differentiation of BCR/ABL – transformed cells regardless of their degree of sensitivity to imatinib (Neviani et al., 2005).
Our previous studies have demonstrated that FSK88, a forskolin derivative, can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages, as shown by enhancing nitroblautetrazolium (NBT) reduction ability and increasing alpha-naphthyl acetate esterase (ANAE) activity. At the same time, FSK88 could decrease the membrane lipid fluidity of the UMR106 cells but had little effect on the normal osteoblastic cells, which possibly indicates that FSK88 could inhibit the proliferation and stimulate the differentiation of cancer cells (Wang et al., 1999). In the present study, we have confirmed that FSK88 induces apoptosis in gastric cancer BGC823 cells and demonstrated that dissipation Δψ
2 Materials and methods
2.1 Materials and cell culture
The human gastric cancer BGC823 cells were obtained from Beijing Institute for Cancer Research. Cells were cultured in RPMI1640 medium (Gibco BRL) supplemented with heat inactivated 10% fetal bovine serum made by Hyclone (Logan, UT, USA), 100
FSK88 was extracted and purified (HPLC pure, ≥98%) by the State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, dissolved in pure grade ethanol. Caspase colorimetric assay kits and caspase-3 inhibitor (z-DEVD-fmk) were purchased from Sigma (St. Louis, MO, USA). The cytochrome c kits were purchased from R&D Systems (Minneapolis, MN, USA). All chemicals were of the highest pure grade available.
2.2 MTT cell viability assay
An MTT (3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) test was performed on cultures 0–72
2.3 Quantitative measurement of apoptosis
Apoptosis was measured by direct determination of nucleosomal DNA fragmentation with the cell death detection ELISA (Roche, Mannheim, Germany), which measures the amount of histone-associated DNA fragments present in the cytosol of apoptotic cells. 1
2.4 Caspase activity assay
After treatment, cells were washed twice with ice-cold PBS and harvested as described previously (Ortiz et al., 2001). The catalytic activity of caspases was measured with their fluorogenic substrate. Briefly, 10
2.5 Assay of mitochondrial membrane potential (Δψ
Alterations in the Δψ
2.6 Cytochrome c release
BGC823 cells were seeded in 2
2.7 RT-PCR analysis for Bcl-2 family gene expression
Total RNA from BGC823 cells was prepared with TRIzol (Invitrogen) according to the manufacturer's instructions. Two micrograms of total RNA from each sample was subjected to reverse transcription using Superscript first strand cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol. PCR reactions were then carried out by mixing 5
Primers used for RT-PCR analysis
2.8 Statistical analysis
Values are expressed as mean
3.1 FSK88 affects BGC823 cell viability in a dose- and time-dependent manner
First we assessed dose- (0.1–30
The dose- and time-dependent effect of FSK88 on cell viability of human gastric cancer BGC823 cells. BGC823 cells were incubated with or without 0.1
3.2 FSK88 induces time-dependent apoptosis in BGC823 cells
The effect of FSK88 on BGC823 cell apoptosis was measured by direct determination of nucleosomal DNA fragmentation, which measures the amount of histone-associated DNA fragments present in the cytosol of apoptotic cells. As shown in Fig. 2, FSK88-induced a moderate to strong apoptotic death in a time-dependent manner, 10
Percentage of apoptotic cells determined by DNA fragmentation. BGC823 cells were treated without (○) or with (●) 10
3.3 Regulation of apoptosis-related Bcl-2 family gene expression in FSK88-treated BGC823 cells
To investigate the molecular mechanism of FSK88-induced apoptosis in BGC823 cells, the gene expression of apoptosis-related Bcl-2 family with or without 10
RT-PCR assay of the effect of FSK88 on Bcl-2 family gene expression in BGC823 cells. The cells were treated with 10
3.4 FSK88 disrupts mitochondrial membrance potential and induces cytochrome c release in BGC823 cells
Mitochondrial intergrity or the loss of Δψ
Measurement of mitochondrial membrane potential and cytochrome c release in human gastric cancer BGC823 cells during the FSK88 treatment. (A) BGC823 cells were treated without (○) or with (●) 10
Cytochrome c release from mitochondrial inter-membranous space into cytosol has been shown to be a key event in the activation of caspase-9 which subsequently initiates a caspase cascade involving caspase-3 (Reed, 1997; Slee et al., 1999), in order to define an upstream event in FSK88-induced apoptosis in BGC823 cells, we also investigated cytochrome c release in cytosolic fraction following FSK88 treatment of cells. Immunoassay of cytosolic fraction of FSK88 (10
These observations suggest an involvement of cytochrome c release from mitochondria as well as a possible disruption of mitochondria in FSK88-induced apoptosis in BGC823 cells.
3.5 FSK88-induced apoptosis is largely mediated via caspase-3 activation
Activation of caspase-3 is a central mechanism of apoptosis in the mitochondria-dependent and death receptor-dependent pathway (Vaux and Korsmeyer, 1999). In the present study, we examined whether caspase-3 activation is involved in the apoptotic process triggered by FSK88.
To investigate whether or not initiator and effector caspases are involved in the apoptotic progression, we measured the activity of caspases-3, -9 and -8 using the synthetic peptide substrates, Ac-DEVD-AMC, AC-LEHD-AFC, and AC-IETD-AMC, respectively. As shown in Fig. 5, caspase-3 activity was detectable after 12
Time course of caspase enzymatic activities. BGC823 cells were treated with 10
Inhibition of caspase-3 activity (B) and attenuation of FSK88-induced cell death (A) by caspase-3 inhibitor (z-DEVD-fmk). Human gastric cancer BGC823 cells were pre-treated with 50
The central finding of the present study is that FSK88, a forskolin derivative, induces apoptotic death of human gastric cancer BGC823 cells through caspase activation involving dissipation of Δψ
It has been suggested that disruption of Δψ
It is known that released cytochrome c binds to Apaf-1 and participates in the activation of caspase-9 (Kluck et al., 1997; Yang et al., 1997; Zhou et al., 1997). The activated caspase-9 activates caspase-3, resulting in the onset of apoptosis. The increases in caspase-3 and caspase-9 are synchronized with the increase in Bax expression and the decrease in Bcl-2 (Tanabe et al., 1998). Our data (Fig. 3) showed that pro-apoptotic Bax and Bad gene expression was up-regulated and anti-apoptotic Bcl-2 gene expression was down-regulated in a time-dependent manner. The time-dependent decrease in the ratios of Bcl-2:Bax and Bcl-xL:Bax showed that the Bcl-2 family genes participated in FSK88-induced apoptosis in BGC823 cells. At the same time, Δψ
As shown in Fig. 5, caspase-9 activity increased about 4.9-fold and caspase-3 activity increased about 6.7-fold after treatment with 10
In conclusion, our study demonstrates that FSK88 is a potential inducer of apoptosis in human BGC823 cells. It is most likely that mitochondrial pathways are involved in FSK88-induced apoptosis in human gastric cancer BGC823 cells. The treatment of human gastric cancer BGC823 cells with FSK88 activates a cell death pathway that regulates the mitochondrial membrane permeability by down-regulation of Bcl-2 and up-regulation of Bax and Bad, triggering cytochrome c release from mitochondria into cytosol. In addition, FSK88 could induce caspase-9 and caspase-3 activation, consequently leading to apoptotic changes, such as DNA fragmentation.
This work was supported by grants from National Major Program For New Drug Research of China (No. 96-906-05-93) and President of Chinese Academy of Sciences special grants (No. 676). We would also like to thank Ms G. J. Li and Mr Q. F. Xue for their excellent technical assistance.
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Received 8 May 2006/25 May 2006; accepted 26 June 2006doi:10.1016/j.cellbi.2006.06.015