HERG K～+ Channel Related Chemosensitivity In Cancer Cells

herg (human ether-a-go-go-related gene) belongs to an evolutionarily conservedmultigenic family of voltage-activated K⁺ channels, the eag (ether a-gò-gò) family. Itis reported that HERG channels may play a prominent role in the control of tumor cellproliferation, differentiation and carcinogenesis. HERG is mainly expressed in the cellmembrane of cardiac myocytes, but absent from other tissues. In some tumors, theexpression of HERG is related with the malignant grade of the tumors. The blockersof HERG channel also inhibit the proliferation and regulate cell invasion of manytypes of cells. Therefore, HERG channel may be a useful target for cancer diagnosisand therapy.The antibacterial drugs share a large part of the clinical drugs that have theblockage effect of herg K⁺ current. In the present study, the aim is to explore theexpression of HERG K⁺ channel and the mechanisms in colon cancer cells.Furthermore, the antitumor effects and biochemical modulators for the drug ofsparfloxacin, a specific antibacterial blocker of HERG channel, were investigated incolon cancer cells.The HERG protein was expressed in the tested human colon cancer cell linesalthough at different degrees, whereas HEK293 cells were as negative control. Inparticular, HCT116 and HT-29 cells expressed HERG protein at higher level, whereasHCT-8 and SW-480 cells expressed it at moderate level. The above results were alsoconfirmed by RT-PCR.To investigate the function of HERG in cells, the strategies of increasing HERGexpression to detect the corresponding alteration in biological features of cancer cellswere used. The expression of HERG protein in herg-transfected HEK293 cells(HEK293/HERG) was remarkably increased than that in wild type HEK293 cells andmock-transfected HEK293 cells (HEK293/MOCK). Results of clonogenic assay and growth curve assay showed that HERG induced the increase of cloning efficiency andcell growth, suggesting that HERG markedly promoted the proliferation of cancercells.Erythromycin, clarithromycin, sparfloxacin, levofloxacin and fluconazole are allantibacterial, which can block herg channel. MTT assay was used to detect theinhibitory effect of these antibacterial on cancer cell proliferation. Results showed thaterythromycin, clarithromycin and sparfloxacin have the inhibitory effect on coloncancer cells. IC₅₀ of levofloxacin and fluconazole is above 300μM and showed nocytotoxicity. The HCT116 cells were the most sensitive to spafloxacin of which IC₅₀is 74.83μM. The HT-29 cells were the most sensitive to erythromycin of whichIC₅₀ is 98.56μM. Therefore, in the next study we selected sparfloxacin anderythromycin to investigate the inhibitory effect of proliferation and mechanism incancer cells.In order to observe the cytotoxic activity of sparfloxacin and erythromycin onnormal cells, we also examined the effect of HERG blockers on the growth andviability of human and mouse normal diploid fibroblasts, which didn't express HERGprotein. In MTT assay, the sparfloxacin inhibited the proliferation of 2BS cells andNIH/3T3 cells with 214.05μM and 232.82μM IC₅₀, respectively. The IC₅₀ values inthe colon cancer cells with HERG over-expression were substantially lower comparedwith that of the diploid cells without HERG expression, also reflecting the fact thatantiprolifetative and cytotoxic activity of sparfloxacin at least partially depended onthe herg expression levels. What's more, the diploid cells retained their viability after1 week of sparfloxacin incubation at concentrations as high as 300μM. HT-29 cells,treated in parallel, lost their viability after 1-2 d at concentrations above 300μM.These results showed the diploid cells could bear high concentrations of sparfloxacin.To investigate the mechanism of sparfloxacin induced inhibition of theproliferation, the alteration of cell cycle distribution and apoptosis were analyzed inthe colon cells. The MTT assay showed sparfloxacin has a dose-dependentantiproliferative and cytotoxic activity on colon cancer cells. The IC₅₀ values inHEK293/HERG cells were lower than that in the wild type HEK293 cells or HEK293/MOCK cells. This result indicated that the sparfloxacin inducedproliferation inhibition of cancer cells was, at least in part, the consequence of theinhibition of HERG channel.Next, the ability of herg blocker—sparfloxacin to induce apoptosis in coloncancer cells was investigated by TUNEL and Annexin V-FITC/PI double staining.After 48 h of exposure to the sparfloxacin, more TUNEL positive cells were seen in300μM sparfloxacin. The sparfloxacin can induce apoptosis of HCT116 cells in adose-dependent manner. The same tendency of induction of apoptosis was confirmedin HCT116 cells by flow cytometry analysis with FITC-Annexin V and PI doublestaining. These results indicated that sparfloxacin could induce apoptotic cell death.The difference of the apoptotic rate between wild-type HEK293 cells andherg-transfected HEK293 cells induced by sparfloxacin was also detected. In thewild-type HEK293 cells, less cells appeared apoptosis, however in HEK293/HERGcells, high concentrations of sparfloxacin induced eminent apoptosis. This experimentconfirmed that apoptosis inducing by sparfloxacin was related with HERG channels.Sparfloxacin led to a dose-dependent decreased the expression of procaspase-3, Bcl-2and NF-κB in correlation with apoptotic cell death in HCT116 cells and HT-29 cellsTo further investigate whether sparfloxacin induced the alteration of cell cycledistribution, flow cytometry analysis was applid and the results showed thatsparfloxacin induced a dose-dependent increase in the fraction of cells in G₂/M phaseof the cell cycle. These data demonstrated that sparfloxacin inhibited cell growththrough a cell cycle arrest in the G₂/M phase.Sparfloxacin treatment at the concentrations of 50μM, 100μM and 200μMdramatically reduced the movement capacity of HCT116 cells. The secretion ofmatrix metalloproteinase MMP-2 and MMP-9 from HCT116 cells was markedlydecreased after 48 h exposure to sparfloxacin as determined by gelatin zymographyassay. In addition, the invasion capacity of tumor cells was significantly decreased inchemo-invasion assay by using the Boyden chamber in vitro. Sparfloxacinsignificantly inhibited the migration of HCT116 cells in a dose-dependent manner.Incubation of exponentially growing HCT116 cells and HT-29 cells with sparfloxacin for 48 h, no significant changes of HERG protein were observed.However, phosphorylations of EGFR and Akt were dramatically suppressed bysparfloxacin. From these results, it was concluded that the down-regulation of NF-κBby sparfloxacin in HCT116 cells is partly mediated via EGFR/Akt pathway.The effects of antitumor drugs combined with sparfloxacin in HCT116 cells wereexamined. The results showed that sparfloxacin in combination with HCPT, CDDP,DOX and MMC had no synergetic effect but with 5-FU, VCR, Taxol had synergeticeffect on the proliferation of HCT116 cells and HT-29 cells. Addition of 10μMsparfloxacin alone on HCT116 and 30μM sparfloxacin alone on HT-29 cells, theinhibitory rates were 89.3% and 84.2%, respectively. However, when addedsimultaneously with different concentrations of 5-FU, VCR and Taxol, the cytotoxityof combined drugs was markedly enhanced in HCT116 cells. Similar results wereobserved in HT-29 cells.The alteration of cell cycle distribution was analyzed when sparfloxacin combinedwith 5-FU in HCT116 cells. Flow cytometry results revealed that no eminent cellcycle arrest was seen in 5μM 5-FU treated group. When sparfloxacin combined with5-FU, the proportion of S and G₂/M phase was elevated compared with control. Theseresults showed sparfloxacin in combination with 5-FU enhanced the ability ofinducing S and G₂/M in arrest.Whether sparfloxacin combined with 5-FU enhanced the ability of inducingapoptosis was also observed. Flow cytometry/PI staining analysis revealed that noeminent apoptosis in 5-FU treated alone. When sparfloxacin combined with 5-FU, theapopototic cell populations of HCT116 cells were elevated compared with control.The results of FITC-Annexin V/PI double staining also showed the same tendency.When combined the 5-FU with 100μM, 200μM SPFX, the early apopototic cellpopulation was up to 28.37% and 33.12%. Sparfloxacin in combination with 5-FUenhanced the ability of inducing apoptosis.Since both sparfloxacin and erythromycin are the HERG blocker, they might takemore significant effect on the cancer cells as attacking the same target. In order toinvestigate this possibility, we combined sparfloxacin and erythromycin as equally mol ratios, named as SPFX-ERM. When administrated the different concentrations ofSPFX-ERM, the mol ratio of sparfloxacin to erythromycin was kept to 1:1, the coloncancer cells were more sensitive to this combinations than respective drug alone.Further, the effects of SPFX-ERM combined with chemocherapy drugs wereexamined in colon cancer cells. Three drug administration methods were comparedthat were: (a) first SPFX, 24 h later ERM added, (b) first ERM, 24 h later SPFXadded or (c) SPFX and ERM were added at the same time. The results showed thatthe chemosensitivity was related with administration method. When combined withdifferent concentrations of 5-FU for 72 h in HCT116 and HT-29 cells, there weresynergistic effects of 10μM SPEX-ERM in combination with 5-FU. These resultssuggest that SPEX-ERM as a compound targeting HERG could potentiate theantitumor activity of 5-FU in colon cancer cells. Another experiment demonstratedthat the drug administration method of SPFX-ERM first and VCR later had eminentlysynergetic effect on the HCT116 and HT-29 cells. But the drug administration methodof VCR first and SPFX-ERM later had no synergetic effect on the HCT116 and HT-29cells.To investigate whether sparfloxacin suppressed tumor proliferation in vivo,antitumor activity of sparfloxacin in the murine hepatoma 22 models in KM mice wasevaluated. The inhibition of tumor growth by sparfloxacin was observed withadministration of i.p. at a dose of 15 mg/kg 5-FU or i.g. at the dose of 200 mg/kg, 400mg/kg and 800 mg/kg every day for ten times. On day 11, the inhibition rate oftreatment of 5-FU was 29.13%. In the combinatory therapy, and 5-FU was given asabove. The inhibition rate with combinatory treatment of 400 mg/kg sparfloxacin and5-FU was 41.08%. We further observed the antitumor effect of SPFX-ERM whengiven i.g. two times every day for ten times, the inhibition rate with treatment of 200mg/kg, 400 mg/kg and 800 mg/kg SPFX-ERM was 37.5%, 64.7% and 88.7%,respectively. In the combinatory therapy, 5-FU was given as 30 mg/kg every other dayfor 5 times. The inhibition rate with combinatory treatment of 400 mg/kg sparfloxacinand 5-FU was 88.7%, whereas the inhibition rate with treatment of 5-FU andSPFX-ERM alone was 63.3% and 64.7%, respectively. And the CDI was 0.85. These results showed that sparfloxacin and SPFX-ERM could enhance the effectiveness of5-FU in vivo.HERG is highly expressed in detected four colon cancer cells and can promote theproliferation of cells. Sparfloxacin, as a herg channel blocker, can inhibit the cellgrowth and induce apoptosis. Sparfloxacin enhanced cytotoxicity combined witherythromycin and antitumor drugs in vitro and in vivo. Sparfloxacin can be abiochemical modulator in treatment of colon cancer with chemotherapy drugs.