Mesothelioma is an aggressive tumor caused by asbestos exposure, the incidence of which is predicted to increase globally. The prognosis of patients with mesothelioma undergoing conventional therapy is poor. Radiation therapy for mesothelioma is of limited use because of the intrinsic radioresistance of tumor cells compared with surrounding normal tissue. Thus, a novel molecular-targeted radiosensitizing agent that enhances the radiosensitivity of mesothelioma cells is required to improve the therapeutic efficacy of radiation therapy. ZDHHC8 knockdown reduces cell survival and induces an impaired G2/M checkpoint after X-irradiation in HEK293 cells. In the present study, we further analyzed the effect of the combination of ZDHHC8 knockdown and X-irradiation and assessed its therapeutic efficacy in mesothelioma models. SiRNA-induced ZDHHC8 knockdown in 211H and H2052 mesothelioma cells significantly reduced cell survival after X-irradiation. In 211H cells treated with ZDHHC8 siRNA and X-irradiation, the G2/M checkpoint was impaired and there was an increase in the number of cells with micronuclei, as well as apoptotic cells, in vitro. In 211H tumor-bearing mice, ZDHHC8 siRNA and X-irradiation significantly suppressed tumor growth, whereas ZDHHC8 siRNA alone did not. Immunohistochemical analysis showed decreased cell proliferation and induction of apoptosis in tumors treated with ZDHHC8 siRNA and X-irradiation, but not with ZDHHC8 siRNA alone. These results suggest that ZDHHC8 knockdown with X-irradiation induces chromosomal instability and apoptosis through the impaired G2/M checkpoint. In conclusion, the combination of ZDHHC8 siRNA and X-irradiation has the potential to improve the therapeutic efficacy of radiation therapy for malignant mesothelioma. (Cancer Sci 2012; 103: 203–209)
Malignant mesothelioma is an aggressive tumor of mesothelial cells,(1,2) most commonly seen in the pleura, followed by the peritoneum, pericardium, and male genitalia. Mesotheliomas are mostly attributed to asbestos exposure, with a lag time of 30–40 years between exposure and the occurrence of disease.(3) Although mesothelioma has been considered a rare tumor, its incidence is anticipated to increase globally(3,4) and peak around 2025 in Japan.(2) Mesothelioma has three main histological subtypes: epithelioid, sarcomatoid, and biphasic. Epithelioid tumors are the most common and are associated with a better prognosis than sarcomatoid and biphasic tumors. Malignant mesothelioma, especially sarcomatoid, is highly lethal; prognosis following therapy very poor, with median survival between 9 and 17 months.(5) Because mesothelioma is mostly diagnosed at an advanced stage, curative surgery is unsuitable and chemotherapy is the preferred treatment strategy.(6) Radiation therapy (RT) is used for symptom palliation and as adjuvant therapy after surgical resection of early stage mesothelioma.(6,7) Moreover, RT is used to alleviate symptoms in advanced-stage mesothelioma, although radiation monotherapy has minimal efficacy in prolonging survival.(6) In addition, RT for mesothelioma is of limited use because of the large target tumor volume, intrinsic tumor radioresistance, and adverse effects on surrounding normal tissue in the irradiated field, such as the heart, liver and lung.(6,8) Thus, a novel molecular-targeted radiosensitizing agent is required to increase the radiosensitivity of mesothelioma cells, thereby improving the efficacy of RT.(9)
The ZDHHC8 gene encodes a palmitoyltransferase belonging to the 23-member family of enzymes that share a conserved cysteine-rich signature catalytic domain (DHHC domain).(10,11) The ZDHHC8 protein is present in the Golgi apparatus and/or endoplasmic reticulum (ER),(11,12) and palmitoylation, a post-translational protein modification, is assumed to occur in the Golgi and ER.(13) In addition, ZDHHC8 is localized in the mitochondria, where it is involved in mitochondria-regulated apoptosis.(14) Functional screening using the genome-wide siRNA library for the human cell line HEK293 identified ZDHHC8 as a novel radiation susceptibility gene;(15) this gene is associated with the G2/M checkpoint response to X-irradiation-induced DNA damage. These findings suggest that ZDHHC8 plays important roles in several biological processes, including cell survival after X-irradiation-induced DNA damage. Thus, it is a candidate molecular target for a radiosensitizing agent, but the therapeutic efficacy of a combination of ZDHHC8 depletion and ionizing irradiation remains unclear.
Therefore, in the present study we further analyzed the effect of a combination of ZDHHC8 knockdown and X-irradiation on cell survival and assessed its therapeutic efficacy in a sarcomatoid mesothelioma model. We demonstrated that ZDHHC8 knockdown with X-irradiation induces chromosomal instability and apoptosis through the impaired G2/M checkpoint in mesothelioma cells, and that combination therapy with ZDHHC8 siRNA and X-irradiation significantly suppresses tumor growth and induces apoptosis in a mouse model.
Materials and Methods
Cell culture and X-irradiation. Human malignant mesothelioma cell lines 211H, H28, H2052, and H2452 and the mesothelial cell line MeT-5A were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI 1640 medium (Sigma, St Louis, MO, USA) supplemented with 5% FCS (JRH Biosciences, Lenexa, KS, USA) and X-irradiated (single dose; 200 kVp) at a rate of 1.07–1.08 Gy/min using the TITAN-320 X-ray generator (GE Healthcare, Little Chalfont, UK).
In vitro siRNA treatment. Two siRNAs for ZDHHC8 (ZDHHC8 #1, 5′-GCUUGCGUUGUGUCUGUUUUA-3′; and ZDHHC8 #2, 5′-GAAACUAUCGCUACUUCUUCC-3′) and negative siRNAs were purchased from RNAi Co. (Tokyo, Japan). Cells were transfected with 50 nM siRNA using the Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA).
Real-time quantitative RT-PCR. First-strand cDNA was synthesized from the cells using the FastLane Cell cDNA kit (Qiagen, Hilden, Germany) 72 h after transfection. Real-time RT-PCR was performed in triplicate using predesigned and preoptimized TaqMan probes to detect ZDHHC8 and 18S rRNA (Applied Biosystems, Foster City, CA, USA). Gene expression levels in each sample were normalized against 18S rRNA expression.
Western blotting. The anti-ZDHHC8 antibody was produced by immunizing rabbits with a synthetic peptide derived from the human ZDHHC8 C-terminal sequence (KKVSGVGGTTYEISV; Scrum, Tokyo, Japan). Cells were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Sigma). Western blotting was performed using anti-ZDHHC8 or anti-β-actin antibody (Sigma) according to standard procedures.
Cell proliferation assay. Seventy-two hours after transfection, cells were X-irradiated with 2, 4, 6, and 8 Gy, seeded in a 96-well plate (2000 cells/well), and cultured for 4 days. A cell proliferation assay was performed using a sulforhodamine B-based Toxicology Assay kit (Sigma).(15,16) The surviving fraction was calculated as the ratio of the absorbance of irradiated cells to that of non-irradiated cells.
Cell cycle analysis. Seventy-two hours after transfection, cells were X-irradiated with 4 Gy. At 4, 8, 16 and 24 h after irradiation, cells were harvested and cell cycle analysis was conducted as described previously.(15)
Micronucleus assay. Seventy-two hours after transfection, cells were X-irradiated with 2 or 4 Gy. Then, 48 h after irradiation, a micronucleus assay was conducted according to standard procedures and at least 1000 cells were observed and scored in each sample.
Apoptosis assay. Seventy-two hours after transfection, cells were X-irradiated with 4 Gy. At 8, 16 and 24 h after irradiation, an apoptosis assay was conducted using the PathScan cleaved caspase-3 sandwich ELISA kit (Cell Signaling Technology). Cleaved caspase-3 levels were normalized against the absorbance of mock control cells at 0 Gy.
In vivo study of a xenografted tumor model. Two million 211H cells were inoculated subcutaneously into BALB/c–nu/nu mice (CLEA Japan, Tokyo, Japan). After 4 days, 50 nM ZDHHC8 or negative siRNA was mixed with atelocollagen (Koken, Tokyo, Japan) and injected around the subcutaneous tumors. Three days after siRNA treatment, the tumors were X-irradiated with 15 Gy (4.5–4.6 Gy/min). Subcutaneous tumor size was measured twice a week using a caliper. Animal experiments were reviewed and approved by the institutional Animal Care and Use Committee of the National Institute of Radiological Sciences.
Histological analysis. Tumors were excised 10 days after X-irradiation and tumor cell proliferation was detected by immunohistochemical staining using anti-Ki-67 mouse monoclonal antibody (DakoCytomation, Glostrup, Denmark). Apoptosis was detected using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Chemicon International, Temecula, CA, USA). Proliferation and apoptosis were quantified as a percentage of positive cells by counting at least 100 tumor cells in four randomly selected fields at ×400 magnification in five sections per treatment.
Statistical analysis. Data are presented as the mean ± SEM. The significance of differences between mean values was evaluated using ANOVA with Dunnett’s multiple comparison test or Student’s t-test. P <0.05 was considered significant.
Effect of siRNA transfection on ZDHHC8 expression. The knockdown efficiency of ZDHHC8 siRNAs on ZDHHC8 mRNA expression was evaluated in 211H cells that had been transfected with ZDHHC8 siRNAs (#1 and #2) or the negative siRNA. Using real-time RT-PCR, we found that compared with the mock control cells, ZDHHC8 mRNA expression was reduced to 14.3 ± 2.7% and 23.3 ± 6.0% in siRNA #1- and #2-transfected cells, respectively (Fig. 1a). In contrast, ZDHHC8 mRNA expression was only slightly decreased in the negative siRNA-transfected cells compared with that in the mock control cells (Fig. 1a). Using western blotting, we found that ZDHHC8 siRNA suppressed ZDHHC8 protein expression, whereas the negative siRNA had no effect on ZDHHC8 expression (Fig. 1b). On the basis of these results and because ZDHHC8 siRNA #1 showed a higher efficiency than ZDHHC8 siRNA #2, we selected ZDHHC8 siRNA#1 for use in subsequent studies.
Moreover, western blotting analysis was performed in an additional three mesothelioma cell lines (H28, H2052 and H2452) and in the mesothelial cell line MeT-5A. All four cell lines expressed ZDHHC8 protein (Fig. 1c). The expression of ZDHHC8 protein was suppressed in H2052 cells transfected with ZDHHC8 siRNA similar to 211H cells. However, in H28, H2452, and MeT-5A cells, although ZDHHC8 siRNA reduced ZDHHC8 protein expression, the reduction was much less than that seen in the 211H and H2052 cells.
ZDHHC8 knockdown suppresses cell proliferation after X-irradiation. The sulforhodamine B-based cell proliferation assay showed that cell survival decreased dose dependently in five cell lines after irradiation (Fig. 2). No differences were observed between the mock control and negative siRNA-transfected cells at all dose levels tested in these cell lines. In the 211H cell line, ZDHHC8 siRNA-transfected cells exhibited significantly reduced cell survival at all dose levels compared with mock control and negative siRNA-transfected cells (P <0.01). At 4 Gy X-irradiation, the fraction of surviving ZDHHC8 siRNA-transfected cells decreased threefold compared with mock control or negative siRNA-transfected cells; this was the greatest difference between ZDHHC8 siRNA-transfected and control groups seen. The cell survival of H2052 cells transfected with ZDHHC8 siRNA was also significantly reduced following 4, 6 and 8 Gy X-irradiation. However, no significant differences were observed for the H28, H2452, and MeT-5A cell lines between mock control or negative siRNA-transfected cells and ZDHHC8 siRNA-transfected cells. On the basis of these results, 211H cells irradiated with 4 Gy were used in subsequent experiments.
ZDHHC8 knockdown induces impaired G2/M checkpoint after X-irradiation. Flow cytometry was used to analyze mock control, negative siRNA-, and ZDHHC8 siRNA-transfected 211H mesothelioma cells, with or without X-irradiation. Under non-irradiated conditions, the cell cycle distribution of ZDHHC8 siRNA-transfected cells was similar to that of the mock control and negative siRNA-transfected cells (Fig. 3a). Using western blot analysis, we confirmed that expression of the G2/M checkpoint-associated proteins Chk1, Chk2, Wee1, cdc25C, Myt1 and cdc2 was unchanged in the mock control, negative siRNA-, and ZDHHC8 siRNA-transfected cells (data not shown). Figure 3(b) shows the temporal change in the cell population in the G0/G1, S, G2/M and sub-G1 phases up to 24 h after X-irradiation. In both the mock control and negative siRNA-transfected cells, 8 h after X-irradiation the G2/M population increased to 61.0 ± 4.9% and 53.3 ± 5.1%, respectively, and the G0/G1 population decreased to 21.2 ± 1.3% and 21.9 ± 0.2%, respectively. These findings indicate that the G2/M checkpoint response was activated after X-irradiation. Conversely, in the ZDHHC8 siRNA-transfected cells, the G2/M population increased slightly to 18.8 ± 4.3% and the G0/G1 population decreased to only 57.3 ± 1.6%. These data suggest that ZDHHC8 knockdown inhibits X-ray-induced G2/M arrest.
ZDHHC8 knockdown increases chromosomal radiosensitivity. Micronucleus formation is considered a reliable marker of ionizing irradiation-induced chromosomal breakage.(17,18) There spontaneous levels of micronucleus formation in mock control, negative siRNA- and ZDHHC8 siRNA-transfected 211H cells were all <1.5%, with no significant differences among the cells (Fig. 4). Although X-irradiation-induced micronucleus formation increased dose dependently in all cells, the increase in micronucleus formation in the ZDHHC8 siRNA-transfected cells after X-irradiation was approximately twofold higher than that in the mock control or negative siRNA-transfected cells (P <0.01).
ZDHHC8 knockdown significantly induces apoptosis after X-irradiation. After X-irradiation, the sub-G1 population among the ZDHHC8 siRNA-transfected 211H cells increased with time, peaking at 16 h. In contrast, there was no increase in sub-G1 population in mock control and negative siRNA-transfected cells (Fig. 3b). Because the sub-G1 population often reflects apoptotic cells, to confirm the contribution of apoptosis to the reduction in cell survival in ZDHHC8 siRNA-transfected cells after X-irradiation, we measured temporal changes in the apoptotic marker cleaved caspase-3 using sandwich ELISA (Fig. 5). Levels of cleaved caspase-3 were higher in ZDHHC8 siRNA-transfected cells than in mock control or negative siRNA-transfected cells (P <0.01). Although levels of cleaved caspase-3 in the mock control cells increased with time after X-irradiation, reaching an increase of 1.4-fold after 24 h, the levels remained significantly lower than those in ZDHHC8 siRNA-transfected cells at all time points investigated (P <0.01). There were no significant changes in cleaved caspase-3 levels in negative siRNA-transfected cells after X-irradiation. In the ZDHHC8 siRNA-transfected cells, levels of activated caspase-3 increased 1.7-fold compared with the non-irradiated cells, peaking at 8 h after X-irradiation (P <0.01).
ZDHHC8 siRNA and X-irradiation enhance the antitumor effect in vivo. The therapeutic efficacy of combination treatment with ZDHHC8 siRNA and X-irradiation was evaluated in a mesothelioma mouse model (Fig. 6a). The volume of 211H tumors treated with negative siRNAs increased in a time-dependent manner, and there were no significant differences between X-irradiated and non-irradiated tumors. The growth of tumors treated with the ZDHHC8 siRNA alone did not differ significantly from that of tumors treated with negative siRNA with or without X-irradiation. However, the combination of ZDHHC8 siRNA and X-irradiation significantly suppressed tumor growth compared with other treatments 10 days after X-irradiation (=17 days after cell inoculation) or later (P <0.01).
Investigations using Ki-67 immunohistochemical and TUNEL staining in xenografted tumors 10 days after X-irradiation revealed that under non-irradiated conditions, the percentage of Ki-67-positive cells was approximately 50% for tumors treated with either negative or ZDHHC8 siRNAs (NS; Fig. 6b,c). However, after X-irradiation, the percentage of Ki-67-positive cells was significantly lower in the ZDHHC8 siRNA-treated group than in the negative siRNA-treated group (28.1 ± 2.8%vs 38.5 ± 4.1%, respectively; P <0.05; Fig. 6b,c). Using TUNEL staining, the percentage of apoptotic cells in tumors treated with negative or ZDHHC8 siRNAs without X-irradiation was 0.5 ± 0.4% and 0.4 ± 0.6%, respectively (NS; Fig. 6d,e). After X-irradiation, the percentage of apoptotic cells in tumors treated with ZDHHC8 siRNAs was significantly higher than in tumors treated with negative siRNAs (9.7 ± 1.5%vs 2.4 ± 1.0%, respectively; P <0.01; Fig. 6d,e).
Malignant mesothelioma, especially the sarcomatoid subtype, is a highly aggressive tumor with a very poor prognosis.(5) One of the major treatments for cancer is RT targeting cellular DNA. However, the efficacy of radiation monotherapy in prolonging survival in patients with mesothelioma is minimal(6) because of the intrinsic radioresistance of the mesothelioma compared with surrounding normal tissues.(6,8) To improve the efficacy of RT, a novel molecular-targeted radiosensitizing agent should be introduced to increase the radiosensitivity of the mesothelioma cells.(9) In the present study, we investigated the potential of ZDHHC8 siRNA as a radiosensitizing agent and evaluated the mechanism underlying its sensitizing effect.
Our cell survival experiments showed that ZDHHC8 siRNA increased the radiosensitivity of 211H and H2052 mesothelioma cells. However, cell survival of H28 and H2452 mesothelioma and MeT-5A mesothelial cells transfected with ZDHHC8 siRNA was not reduced compared with the survival of cells transfected with mock control or negative siRNA. Because ZDHHC8 siRNA did not completely suppress protein expression in those cell lines, these results may be due to insufficient knockdown efficiency. To further investigate the radiosensitizing mechanism caused by ZDHHC8 knockdown, we analyzed cell cycle progression in the ZDHHC8 siRNA-transfected 211H cells, with or without ionizing irradiation. Cell cycle checkpoint activation is known to play a key role in DNA repair and cell survival following X-irradiation-induced DNA damage. Cell cycle arrest provides irradiated cells the time to repair DNA damage(19,20) and the G2/M transition phase is one of main checkpoints for cell fate determination after X-irradiation.(21) In the present study, G2/M arrest was not observed in ZDHHC8 siRNA-transfected 211H mesothelioma cells after X-irradiation; this finding is consistent with our previous study in HEK293 cells and the results suggest that ZDHHC8 downregulation induces impaired G2/M arrest after X-irradiation.(15)
The impaired G2/M checkpoint contributes to chromosome instability in irradiated cells.(22) A micronucleus is formed during cell division when the nuclear envelope is reconstituted around chromosome fragments, resulting in an inability to repair ionizing irradiation-induced DNA double-strand breaks. Therefore, micronucleus formation is considered a reliable marker for ionizing irradiation-induced chromosomal damage.(17,18) Cells with a micronucleus typically cannot reproduce because of their inability to pass through the cell cycle checkpoints. As shown in the present study, micronucleus formation in the ZDHHC8 siRNA-transfected cells increased approximately twofold compared with that in mock control or negative siRNA-transfected cells after X-irradiation with 2 and 4 Gy. These findings suggest that the imperfect G2/M checkpoint induced by ZDHHC8 knockdown caused the accumulation of unrepaired DNA damage after irradiation, and then enhanced chromosome instability and inhibited cell proliferation, leading to an increase in the radiation sensitivity of mesothelioma cells.
Accumulation of unrepaired damaged DNA possibly induces apoptosis,(23) so we analyzed the involvement of apoptosis in the radiosensitizing effect of ZDHHC8 siRNA. The sub-G1 population and levels of cleaved caspase-3 increased after X-irradiation of ZDHHC8-knockdown cells, but not in mock control cells, suggesting that ZDHHC8 depletion induced apoptosis after X-irradiation. There was a difference in the time of the peak effect on sub-G1 population and activated caspase-3 levels in ZDHHC8 siRNA-transfected cells after X-irradiation. These differences could indicate that apoptotic proceeds in cells in which caspase-3 is activated (early in the process of apoptosis) and that the sub-G1 population is observed in the final stage. Although ZDHHC8 is involved in mitochondrial-regulated apoptosis when it is overexpressed,(14) in case of ZDHHC8 downregulation, apoptosis seems to be induced by a lack of double-strand break repair through the impaired G2/M checkpoint rather than by mitochondrial dysfunction. Ionizing irradiation causes not only apoptosis, but also other types of cell death, such as necrosis and autophagy.(24,25) Therefore, other types of cell death could also be induced in the ZDHHC8 siRNA-transfected cells after X-irradiation, which needs to be clarified in further studies.
We evaluated the therapeutic efficacy of ZDHHC8-targeted radiosensitization by administering RT to a sarcomatoid mesothelioma mouse model. The growth of xenografted tumors was significantly suppressed with the combination of ZDHHC8 siRNA and X-irradiation compared with the combination of negative siRNA and X-irradiation. Histological analysis of xenografted tumors showed that the combination of ZDHHC8 siRNA and X-irradiation decreased cell proliferation and increased apoptosis, which was also observed in vitro. Although cell cycle distribution after X-irradiation could not be analyzed in mouse tumor models, it can be speculated that induction of apoptosis in vivo could be the result of an impaired G2/M checkpoint, similar to the findings in vitro. These findings suggest that the combination of ZDHHC8 siRNA and RT could be more effective in treating mesothelioma compared with radiation monotherapy.
In the present study, we demonstrated that siRNA targeted at ZDHHC8 could effectively enhance the radiosensitivity of mesothelioma cells. For advanced clinical application of this technique, development of specific and effective carriers of siRNA to target tumor tissue is essential. Recently, it was reported that nanoparticles, such as liposomes and nanomicelles, containing siRNAs successfully inhibited tumor growth in animal models,(26,27) and clinical trials of liposome–siRNA complexes for cancer therapy have been conducted.(27) Nanoparticles, with a diameter of 100–200 nm, are known to accumulate more in tumors than in normal tissues because of enhanced permeability and retention effects.(26) Thus, nanotechnology-based ZDHHC8 siRNA delivery systems, such as a liposome–siRNA complex, could further enhance the radiosensitivity of mesotheliomas while reducing adverse side effect in patients treated with the combination therapy. Furthermore, for malignant pleural mesothelioma, intrapleural administration of siRNA–nanoparticles could be more effective and further reduce the adverse effects on normal organs compared with systemic administration.
Thus, ZDHHC8 depletion significantly enhanced the radiosensitivity of human mesothelioma cells, both in vitro and in vivo. Radiosensitization caused chromosomal instability and cell death, including apoptosis because of a lack of repair of double-strand breaks through the impaired G2/M checkpoint. Our findings reveal that ZDHHC8 siRNA is an effective radiosensitizing agent that improves RT efficacy in patients with human malignant mesothelioma.
The authors are thankful to Dr Yasushi Kataoka (National Institute of Radiological Sciences, Chiba, Japan) for valuable discussions of the manuscript.