Expression and significance of HERG protein in gastric cancer

[Cancer Biology & Therapy 7:1, 45-50; 1 January 2008]; ©2008 Landes Bioscience

Research Paper

Expression and significance of HERG protein in gastric cancer Xiao-Dong Shao1,2,*, Kai-Chun Wu2, Xiao-Zhong Guo1, Man-Jiang Xie3, Jing Zhang2, Dai-Ming Fan2 1Department

of Gastroenterology; Shenyang General Hospital; Shenyang, China; 2State Key Laboratory of Cancer Biology, Department of Gastroenterology; Xijing Hospital; Fourth Military Medical University; Xi’an, China; 3School of Aerospace Medicine; Fourth Military Medical University; Xi’an, China

Key words: gastric cancer, human ether-a-go-go-related gene (herg), proliferation, apoptosis

Objectives: Our previous studies showed that delayed rectifier potassium currents existed in human gastric cancer cells and the currents were related to the growth of gastric cancer cells. Human ether‑a‑go‑go‑related gene (herg) encoding a subunit of delayed rectifier potassium channel has been indicated with involvement in tumor cell growth and death. The purpose of the present study is to investigate the expression of HERG protein in gastric cancer tissue and cells; analyze the relationship between the expression of HERG protein and the clinicopathological characteristics of patients with gastric cancer; explore the effects of HERG protein on biological behaviours of gastric cancer cells. Results: HERG protein was exclusively expressed in gastric cancer cells. The expression of HERG protein was associated with tumor differentiation, TNM stage and lymph node involvement of gastric cancer. Silencing HERG protein could eliminate the HERG currents and inhibit proliferation, clone formation, invasiveness and tumorigenicity of gastric cancer cells. Reducing HERG protein could also inhibit gastric cancer cells entering S phase from G1 phase and induce apoptosis of gastric cancer cells. Methods: The expression of HERG protein in gastric cancer tissues and cells was measured by immunohistochemistry and Western blot, respectively. Reduction of HERG protein was carried out by siRNA technology. The proliferation, ability of clone forma‑ tion, cell cycle, apoptosis and invasive ability of gastric cancer cells were evaluated by MTT assay, clone formation assay, flow cytom‑ etry and cell invasion assay. Tumor growth in nude mice was to be used to access the tumorigenicity of gastric cancer cells and HERG currents were recorded by patch‑clamp. Conclusion: HERG protein is involved in carcinogenesis of gastric cancer and is a potential therapeutic target of gastric cancer.

Introduction herg belongs to an evolutionary conserved multigene family of voltage‑activated outward rectifier potassium channels, the eag family. In the heart, the rapid delayed rectifier potassium current (Ikr), the *Correspondence to: Xiao-Dong Shao; Department of Gastroenterology; Shenyang General Hospital; Shenyang 110016 China; Tel.: 86.24.23051111; Fax: 86.24.23056230; Email: [email protected] Submitted: 07/29/07; Revised: 10/07/07; Accepted: 10/08/07 Previously published online as a Cancer Biology & Therapy E-publication: www.landesbioscience.com/journals/cbt/article/5126

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physiological counterpart of herg, undergoes remarkable develop‑ ment patterns, predominating in the fetal heart and dissipating in the adult. When the adult cardiac cells become dedifferentiated or cancerous, such as AT‑1 and HL‑1 (murine atrial tumor cell lines) cells, Ikr regains its predominance among the potassium channels.1,2 HERG currents are transiently expressed at very early stages in neural crest neuron developments, but disappearing at later stages to be substituted by inward rectifier‑like currents. Recent studies showed that HERG protein is preferentially expressed in tumor cell lines of different histogenesis.3‑5 It was believed that in tumor cells, HERG current is responsible for maintaining substantially depolarized resting potentials, a feature in cancer cells. Some researchers proposed that HERG channels were involved in the regulation of tumor cell growth and death. Our group had found delayed rectifier potassium currents in human gastric cancer SGC7901 cell line and the currents were related to the growth of gastric cancer cell.6 Recently we reported that the blocker of HERG channel could inhibit proliferation and induce apoptosis of gastric cancer cells.7 Based on these findings, we designed the present study to evaluate HERG expression in gastric cancer cells and investigate the role of HERG protein in regulating proliferation and apoptosis of gastric cancer cells.

Results Expression of HERG protein in gastric cancer cells and tissues. To investigate the expression of HERG protein in gastric cancer cell lines, Western blot was carried out. Four gastric cancer cell lines (SGC7901, AGS, MGC803 and MKN45) and immortalized gastric mucosa epithelial cell line GES were analyzed. The result showed that HERG protein was exclusively expressed in gastric cancer cells when compared with normal gastric epithelial cells (Fig. 1). The expression of HERG protein was studied by histochemistry with 45 gastric cancers and corresponding normal mucosal specimens. The result revealed that HERG protein was expressed in the gastric cancer cells, but negative expression in gastric mucosal tissues (Fig. 2). Further analysis of the clinicopathological characteristics of the 45 gastric cancer specimens showed that the index of HERG staining in poorly differentiated gastric cancer samples was significantly higher than that in well and moderately differentiated samples. With respect to the TNM stage, the index of HERG staining was much higher in samples of III + IV than that of I + II stage (p < 0.05). There was a statistically significant difference in the HERG staining index between tumors with lymph node involvement and those without lymph node involvement (p < 0.05) (Table 1).

Cancer Biology & Therapy

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HERG and gastric cancer

Figure 1. Detect HERG protein expression in cells by Western blot. Lane 1, SGC7901; lane 2, AGS; lane 3, MGC803; lane 4, MKN45; lane 5, GES.

Table 1

Clinicopathological association of HERG expression in patients with gastric cancer

Figure 2. Immunohistochemical staining of HERG protein in gastric tissues. (A) Well‑differentiated, (B) moderately‑differentiated, (C) poorly‑differentiated gastric cancer tissues showed HERG immunosignals, but (D) normal gastric tissues exhibited negative HERG immunostaining.

a < 0.05 was considered as statistically significant.

Effect of HERG protein on proliferation of gastric cancer cells. To explore whether or not HERG protein might affect the prolifera‑ tion of gastric cancer cells, we used siRNA in an attempt to reduce the expression of HERG protein. siRNA was transfected into SGC7901 cells and stable transfectants were selected by G418 and limiting dilution. As shown in Figure 3A, the siRNA1 can effectively reduce the endogenous level of HERG protein with the inhibitory rate of nearly 90%, compared with siRNA2. So we selected the gastric cancer cells transfected with siRNA1 (SGC7901s1) and pSuppressor vector alone (SGC7901v) to be used for the following experiments. There are specific HERG currents in SGC7901 and SGC7901v cells, but we had not detected HERG currents in SGC7901s1 cells (Fig. 3B). These results meant that siRNA1 effectively inhibited HERG protein and currents in SGC7901 cells. As shown in Figure 4, the growth rate of SGC7901s1 was significantly slower than that of SGC7901 and SGC7901v. After 46

incubation for two weeks, SGC7901s1 presented reduced ability to grow into clones, compared with SGC7901 and SGC7901v (Fig. 5). We also observed the effect of HERG protein on gastric cancer cells by Flow cytometry. The SGC7901s1 cells tended to accumulate in G1 phase, compared with SGC7901 and SGC7901v and the PI of SGC7901s1 was lower than that of SGC7901 and SGC7901v (Fig. 6). These results indicated that HERG protein might partici‑ pate in the regulation of proliferation in SGC7901 cells through affecting cell cycle progression. Influence of HERG protein on invasiveness and tumorigenicity of gastric cancer cells. The role of HERG protein in the invasive‑ ness of gastric cancer cells was studied by analyzing the effect of HERG protein on cell migration through Matrigel‑coated porous membrane inserted into chambers. The number of cell migrating through membrane in SGC7901s1 was significantly less than that in SGC7901v and SGC7901 (p < 0.05) (Fig. 7A), indicating reduction of HERG protein could inhibit invasiveness of gastric cancer cells. The parental cells and the transfected cells were further examined for tumorigenicity by injecting cells subcutaneously into BALB/c nu/nu mice. Tumorigenesis was found to be dramatically lower in SGC7901s1 cells than in SGC7901 and SGC7901v (Fig. 7B), suggesting that the expression of HERG protein is involved in the growth of gastric cancer cells in vivo. Impact of HERG on apoptosis of gastric cancer cells. As shown in Figure 8, Flow cytometry analysis with FITC‑Annexin V and PI double staining revealed that the apoptotic cell population with FITC‑Annexin V+ dramatically increased in SGC7901s1, compared with SGC7901 and SGC7901v.

Discussion Several studies showed that voltage‑gated potassium channels (Kv) play an important role in the proliferation of normal as well as transformed cells.9‑11 Some kinds of cancer cells express a diversity of ion‑channel types and potassium channel‑blockers have been reported to inhibit the proliferation of cancer cells.12,13 Proliferation of DLD‑1 colon carcinoma cells was reduced in the presence of 4‑aminopyridine, a Kv inhibitor.14 In particular, the ether‑a‑go‑go gene (eag) family of outward rectifier potassium channels with its member eag and herg appears to be deeply involved in carcinogenesis.


2008; Vol. 7 Issue 1


Figure 3. Inhibition of HERG protein and current by siRNA. (A) Analysis of HERG protein expression by Western blot. Lane 1, SGC7901; lane 2, SGC7901v; lane 3, SGC7901s1; lane 4, SGC7901s2. (B) Detect HERG current in gastric cancer cells. Current was elicited by 400‑ms‑long hyperpolarizing pulse between +20 and ‑120 mV (in 20 mV increments) from a holding potential of 0 mV. Trace 1, current from a voltage‑clamped SGC7901 cell under control conditions; trace 2, after superfusing with the selective HERG blocker, cisapride (100 nM); trace 3, isolated HERG current by subtracting the trace 2 from the trace 1; trace 4, HERG current detected in SGC7901v; trace 5, no detectable HERG current in SGC7901s1.

The expression of these channels seems to be a selective advantage for tumor cells and the therapeutic potential exists for the eag family.15 Antisense reduction of eag mRNA expression decreased the prolifera‑ tion of several tumor cell lines. The selective HERG channel blocker reduced proliferation of human leukemia CEM, U937 and K562 cell lines.16 Our findings revealed that HERG protein was exclu‑ sively expressed in gastric cancer cells compared with gastric mucosa epithelial cells. Clinicopathological analysis revealed that expression of HERG protein was correlated with tumor differentiation and TNM stage of gastric cancer. Knocking down HERG protein could eliminate the HERG currents in gastric cancer cells and inhibit the growth, clone formation and tumorigenicity of human gastric cancer cells, supporting that Kv are involved in proliferation of tumor cell. The potassium channel activity plays an important role during cell cycle progression. During progression from the G1 to S phase, many cells undergo changes in membrane potential, cell volume, cytoplasmic pH and ion content,17,18 that can arise from different potassium channel expression. Potassium channels differentially regulate the electrical potential of the plasma membrane (Vm) during proliferation. It has been demonstrated that in tumor cells the value of Vm is clamped to rather depolarized values by channels belonging to the herg family. Activation of potassium channels might be required for the passage of cells through a specific phase of the mitotic cycle.19 In different models, an increase in potassium channel expression and activity occurs at the G1/S boundary and such an increase is necessary for cells to traverse the cell cycle. In present study, flow cytometric analysis showed that SGC 7901 cells trans‑ fected with HERG‑siRNA seemed to be prevented from entering S phase of cell cycle and this finding was consistent with previous reports. HERG channels play an important role in the regulating progress of cell cycle by influencing the rest membrane potential of tumor cells. So we speculated that silencing HERG protein might inhibit growth of tumor cells by preventing from entering S phase of cell cycle. www.landesbioscience.com

Reduced apoptosis is a crucial event in carcinogenesis induced by different pathways. Some studies revealed that potassium channel blockers can induce apoptosis of tumor cells.20 The role of HERG protein in cell death has not yet been investigated, but there were some studies about Kv channel involvement in apoptosis. Fas recep‑ tor‑mediated inhibition of Kv1.3 channels may influence apoptosis in the human leukemic T‑cell line Jurkat and two apoptotic proteins, Reaper and Grim, are thought to facilitate apoptosis by stably blocking Kv channels.21,22 In both studies, the interpretation was that inhibiting potassium channels depolarizes the cells and initiates a caspase‑dependent apoptotic pathway. Some authors attributed the effect of potassium channels on apoptosis to the changes of osmolarity and cell volume induced by the potassium currents.23‑25 In cancer cells, by depolarizing the resting potential, HERG current may affect cellular processes such as calcium entry, which in turn may affect apoptosis. In the present study, FITC‑Annexin V and PI double staining showed that there was a substantial apoptotic cell population in SGC7901 cells transfected with siRNA targeting HERG compared with control group. Although the mechanism was not fully understood, the effect of HERG protein on apoptosis of gastric cancer cells suggested that HERG channel plays important role in carcinogenesis of gastric cancer. Moreover, clinicopathological characteristics analysis indicated that HERG protein expression was correlated with lymph node involvement of gastric cancer and subsequent invasion assay showed that inhibiting HERG protein expression did reduce the invasiveness of gastric cancer cells. Similar finding has been recently reported in colon cancer, but its mechanism is not clear.26 The finding of the relationship between HERG protein and integrin may provide new clue for further study.27 In summary, reducing HERG protein expression can inhibit in vitro and in vivo proliferation of gastric cancer cells and induce apoptosis of gastric cancer cells, indicating its potential for treatment of gastric cancer. Further studies are needed to explore the relevant mechanisms and the value of therapeutic application of HERG blockers.

Materials and Methods Cell culture. The human gastric cancer SGC7901, AGS, MGC803 and MKN45 cell lines and human immortalized gastric mucosa epithelial GES cell line were cultured in RPMI1640 medium containing 10% heat‑inactivated fetal bovine serum and incubated at 37°C in a humidified atmosphere with 5% CO2. Tissue collection. A total of 45 pairs of primary gastric cancer tissue and corresponding normal tissue (>5 cm away from the margin of cancer) samples were collected from surgery in Xijing hospital from 2000 to 2002. Of these patients, 26 were male and 19 were female. There were 13 patients with well differentiated tumors, 14 with moderately differentiated tumors and 18 with poorly differenti‑ ated tumors. With respect to macroscopic types of gastric cancer, one tumor was classified as Type 0, 7 as Type 1, 16 as Type 2, 15 as Type 3 and 6 as Type 4 according to the Borrmann’s classification. Regional nodal involvement was detected in 26 patients. No distant metastases were noted. Four patients were classified as pathological stage I, 14 as II, 20 as III, 7 as IV according to TNM classification. The samples were fixed in 10% formaldehyde, dehydrated and embedded in paraffin. Five mm‑thick sections were sliced.


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HERG and gastric center

Figure 4. Growth curve of gastric cancer cells. The value shown is the mean of three determinations.

Immunohistochemistry. Immunohistochemical SP staining method was used in our experiment. Briefly, the tissue sections were routinely dewaxed and hydrated, then treated with 3% peroxide for ten minutes. Antigen restoration was carried out by heating in citrate buffer. The sections were blocked with normal rabbit serum and followed by incubating overnight with anti‑HERG polyclonal antibody (Santa Cruz Bio) at 4°C. Then the sections were incubated with rabbit anti‑goat IgG for 30 minutes at 37°C. After washed with PBS, the sections were treated with DAB at room temperature for four minutes and counterstained with haematoxylin. Negative control was designed with PBS instead of antibody. All sections were examined independently by two observers with respect to the various histopathological characters and specific immu‑ noreactivity (IR). The staining was semiquantitatively evaluated by assigning a score for the intensity of immunoreactivity and for the proportion of cells positively stained (PROP). The product of these two values was used for calculation of the overall IR score (TS), as described by Vandeputte.8 The intensity of the immunoreactivity (intensity score) was stratified into four categories: 0, no IR; 1, weak (+) IR; 2, moderate (++) IR; and 3, strong (+++) IR. The proportion of positive cells (PROP) was classified into four groups: 0, no tumor cells exhibiting IR; 0.33, 67‑100% of the tumor cells exhibiting IR. Western blot. Cell proteins were separated by SDS polyacryl‑ amide gel electrophoresis and transferred to a nitrocellulose sheet. After transfer, the membranes were blocked for four hours at room temperature with TBST containing 5% BSA and then incubated

Figure 5. Ability of clone formation of gastric cancer cells. The data represent means ± SD of three independent experiments.

overnight at 4°C with anti‑HERG polyclonal antibody diluted 1:1000 in TBST. Membranes were then washed three times with TBST and incubated with anti‑goat peroxidase‑conjugate secondary antibodies for one hour at room temperature. After three washes with TBST, the immunoreactivity was determined by a chemiluminescent reation (ECL). b‑actin served as an internal control. Plasmid construction and transfection. Two pairs of hairpin siRNA oligos for herg were designed according to software online (Dharmacon siRNA Design Center). Target sequences were aligned to the human genome database in a BLAST search to ensure that the chosen sequences were not highly homologous with those of other genes. For oligo‑1, S: 5'‑tcg agg agc cgt aag ttc atc atc ttc aag aga gat gat gaa ctt acg gct ctt ttt t‑3', AS: 3'‑cct cgg cat tca agt agt aga agt tct ctc tac tac ttg aat gcc gag aaa aaa gat c‑5'; for oligo‑2, S: 5'‑tcg agg gag cgc aaa gtg gaa atc ttc aag aga gat ttc cac ttt gcg ctc ctt ttt t‑3', AS: 3'‑ccc tcg cgt ttc acc ttt aga agt tct ctc taa agg tga aac gcg agg aaa aaa gat c‑5'. For annealing to form DNA duplexes, 100 mM of S and AS oligos each was used. The duplexes were diluted and then ligated with pSuppressor vector (Imgenex) which was previously digested by the SalI/XbaI restriction enzyme. The products were transformed into DH5a competent cells. Ampicillin‑resistant colonies were chosen, identified by restriction digestion, and further confirmed by DNA sequencing. SGC7901 cells were planted in six‑well plates and cultured in drug‑free medium. At 90–95% confluence, cells were washed

Figure 6. The cell cycle distribution and the proliferation indexes (PI) of gastric cancer cells.

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Figure 8. Apoptosis of gastric cancer cells evaluated by flow cytometry. Apoptotic cells were distributed in right lower quadrant.

Figure 7. The effect of HERG protein on invasiveness and tumorigenicity of gastric cancer cells. (A) Measurement of invasiveness of gastric cancer cells by transwell assay. (B) Tumorigenicity of gastric cancer cells in BALB/c nu/nu mice. The volumes of tumors were monitored at the indicated time.

twice with PBS and grown in 2 ml DMEM without antibiotics. Using Lipofectamine 2000 reagent (Invitrogen), 2 mg of pSuppres‑ sor‑HERG siRNA plasmids was transfected into SGC7901cells according to the manufacturer’s instructions. The cells transfected with pSuppressor vector alone or pcDNA3.1 vector alone served as negative control. Forty‑eight hours later, cells were placed in growth medium containing G418 (GIBCO) for clone selection. The expres‑ sion levels of HERG protein in G418‑resistant clones were evaluated by Western blot analysis. Patch‑clamp recordings. Patch‑clamp recordings were performed at room temperature with an amplifier Axonpatch 1‑D (Axon Instruments), replacing the Petri dish every 30 minutes. The whole cell configuration of the patch‑clamp technique was employed, using pipettes whose resistance was in the range of 3‑5MW. Extracellular solutions were delivered through a remote‑controlled linear posi‑ tioner placed near the cell under study. The extracellular solution contained (mM): NaCl 95, KCl 40, CaCl2 2, Hepes 10, glucose 5, pH 7.4. The standard pipette solution contained (mM): KCl 130, NaCl 10, MgCl2 2, CaCl2 2, EGTA 10, Hepes 10, pH 7.4. Gigaseal resistance was in the range of 3–20 GW. Whole cell currents were filtered at 5 kHz. Input resistance of the cells was in the range of 2‑6 GW. The specific HERG blocker, cisapride (Xi’an‑Janssen), was used at the final concentration of 100 nM. MTT Assay. Briefly, cells in the logarithmic growth phase were harvested and seeded in 96‑well plates (Costar). The cell number was diluted to 5000/well in 200 microliters of RPMI1640 medium. www.landesbioscience.com

Twenty microliters of MTT (Sigma) was added to each well of the cell culture 4 hour before termination of the culture. One hundred and fifty microliters of dimethyl sulfoxide was added to each well at the end of the culture and the plate was agitated for 10 minutes. The absorbency at 490 nm was read by a BIOHIT BP800 plate reader (BIOHIT PIc). Each assay was performed in triplicate. Clone formation assay. Cells were seeded into dishes (100 per dish) and cultured in RPMI 1640 medium for two weeks to allow clone formation. Clones were fixed in 70% ethanol, stained with hematoxylin and counted. Clones of >50 cells were counted at low magnification. The experiments were repeated at least three times. Cell cycle distribution. Gastric cancer cells were harvested by centrifugation and washed with PBS. The cells were fixed with ice‑cold 75% ethanol at 4°C for 18 hours. The fixed cell suspensions were washed with PBS, and then treated with 800 ml 50 mg/L PI dye and 50 mg/L RNase for 30 minutes in the dark. Samples were run through a FACScan (ELITE). Results were presented as the number of cells versus the amount of DNA as indicated by the intensity of fluorescence. The proliferous indexes (PI) were calculated: PI = (S + G2)/(S + G2 + G1). Flow cytometry with FITC‑annexin V and propidium iodide (PI) double staining. After washing twice with PBS, 1 x 106 gastric cancer cells were resuspended in binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2). FITC‑Annexin V (Boehringer Mannheim) was added at a final concentration of 1 mg/ml Annexin V. Then 10 mg/ml PI was added. The mixture was incubated for 10 minutes in the dark at room temperature and then measured by a FACScan using Cell Quest software (Becton Dickinson). Cell invasion assay. One hundred ml of gastric cancer cells suspen‑ sion (1 x 105) were inoculated into the upper well of a chamber (Costar) coated with Matrigel (B&D). After 24 hours of incubation at 37°C, the filters were fixed with 95% ethanol and then stained with hematoxylin and eosin. The cells on the upper surface of the filters were removed by wiping them with a cotton swab. The cells that had invaded through Matrigel and reached to the reverse side were counted under a microscope in five predetermined fields at a magnification of ×400. Each assay was performed in triplicate. Tumor growth in nude mice. The logarithmically growing cells were trypsinized and resuspended in D’Hanks solution, and 5 x 106 cells in 0.2 ml were injected subcutaneously into the left flank of 4‑week‑old female BALB/c nu/nu mice. Experimental and control groups had at least three mice each. The volume of tumor was calcu‑ lated as volume = (length x width2) x 0.5. Statistical analysis. Results are expressed as mean ± standard deviation (SD). Statistical analyses were performed with SPSS 11.0 statistical software. Independent‑Samples t‑test and one‑way analysis of variance (ANOVA) were adopted. Significance was defined as p < 0.05.


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Acknowledgements

This study was supported by grants from the Chinese National Foundation of Natural Science (No. 30170422 and No. 30400204). References

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