Dual role of acetaminophen in promoting hepatoma cells
apoptosis and kidney fibroblasts proliferation
YUNG-LUEN YU1,2,3#, GIOU-TENG YIANG4,5#, PEI-LUN CHOU6,7, HSU-HUNG TSENG8, TSAI-KUN WU2,9, YU-TING HUNG10, PEI-SHIUAN LIN10,
SHU-YU LIN10, HSIAO-CHUN LIU11, WEI-JUNG CHANG1, CHYOU-WEI
WEI10*
1. Graduate Institute of Cancer Biology, and Center for Molecular Medicine, China Medical University, Taichung 404, Taiwan
2. The Ph.D. program for Cancer Biology and Drug Discovery, China Medical
University, Taichung 404, Taiwan
3. Department of Biotechnology, Asia University, Taichung 413, Taiwan 4. Department of Emergency Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu
Chi Medical Foundation, New Taipei 231, Taiwan
5. Department of Emergency Medicine, School of Medicine, Tzu Chi University, Hualien 970, Taiwan
6. Division of Allergy-Immunology-Rheumatology, Department of Internal Medicine, Saint Mary’s Hospital Luodong , Yilan 265, Taiwan
7. Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
8. Division of General Surgery, Taichung Hospital, Ministry of Health and Welfare,Taichung 403, Taiwan
9. Division of Renal Medicine, Tungs' Taichung Metroharbor Hospital,
Taichung 435, Taiwan
10. Department of Nutrition, Master Program of Biomedical Nutrition, Hungkuang University, Shalu, Taichung 433, Taiwan
11. Department of Nursing, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei 231, Taiwan
#These authors contributed equally to this work
*Correspondence to: Dr. Chyou-Wei Wei, Department of Nutrition, Master
Program of Biomedical Nutrition, Hungkuang University, Shalu, Taichung 433, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Taiwan
E-mail: [email protected]
Running title: APAP increase H2O2 level and activate caspases Key words: Acetaminophen, kidney tubular cell, hepatoma, fibroblasts 35 36 37 38 39 40
Abstract: Acetaminophen (APAP), under therapeutic dose, is a safe analgesic and antipyretic drug widely used in clinical cases. However, high-dose APAP (overdose) can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high-dose APAP-induced acute liver and kidney injury. So far, few studies have investigated therapeutic dose (1/10 high-dose) or low-dose (1/100 high-dose) effects on APAP-treated cells. The aim of this study is to understand the cellular effects of therapeutic or low-dose APAP on hepatoma cells and kidney fibroblasts. In this study, as expected, high-dose inhibits cell survival, and therapeutic dose or low-dose APAP does not inhibit cell survival on normal kidney tubular epithelial cells. However, APAP can induce H2O2 level increases and activate caspase-9/-3 cascades resulting in cell apoptosis on hepatoma under therapeutic dose treatments. Moreover, it is interesting that APAP promotes cell proliferation on fibroblasts even under low-dose treatment. This study demonstrates that different cellular effects can be induced with different APAP dose treatments. Our studies indicate that therapeutic dose APAP treatment may exert an anti-tumor activity on hepatoma and low-dose APAP treatment may be harmful to fibrosis patients, resulting in fibrosis deterioration.
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Introduction
Acetaminophen (APAP), also called paracetamol, is a safe analgesic and antipyretic agent at therapeutic dose. It has been applied widely in clinical cases . In general, APAP overdose of 10-15 gram can cause serious toxicity and is harmful to liver and kidneys . APAP is easily available and cheap, and patients may use an overdose of APAP. Therefore, APAP is the most common agent to cause self-poisoning in many countries . In order to study APAP overdose-induced liver and acute kidney damage, many animal models and cell models have been established. These models showed that high-dose APAP of 300-2500 mg/kg can cause hepatotoxicity and nephrotoxicity in experimental animals , and high-dose APAP (above 5 mM) can induce cell cytotoxicity on kidney and liver cells . Although previous studies have shown that APAP can induce apoptosis or necrosis on different cell models , these studies have demonstrated that high-dose APAP can increase oxidative stress, decrease glutathione levels, and activate MAPK signal pathways, resulting in cell cytotoxicity .
Recently, most studies have significantly noticed that high-dose APAP causes liver and kidney failure. However, some studies have reported that high-dose APAP also exerted anti-cancer activities. These studies showed that APAP can induce cell cytotoxicity on neuroblastoma (SH-SY5Y cells), hepatoma (HuH7 cells), and breast cancer (FM3A cells) . These studies also demonstrated that APAP-induced cell death is related to NFKB, Bcl2 family, or glycogen synthase kinase-3 on different tumor cells. In addition, APAP can enhance chemotherapeutic drug-mediated anti-cancer activities on neuroblastoma, leukemia, and ovarian carcinoma . According to the above studies, APAP can induce different cytotoxic mechanisms among different liver cells, kidney cells, and tumor cells . Up to now, most studies have been devoted to investigating the mechanisms of APAP-induced cytotoxicity and how to prevent high-dose APAP poisoning of the liver and kidneys. However, whether APAP can enhance cell proliferation still remains unclear.
Kidney tubular epithelial cell damage can induce renal failure . Moreover, kidney fibrosis, via fibroblast proliferation, can also cause renal failure . Therefore, both kidney tubular cell damage and fibroblast proliferation can cause kidney dysfunction. Recently, high-dose APAP-induced nephrotoxicity was noticed and investigated . These studies found that high-dose APAP can induce 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
kidney tubular cell death on animal and cell models. In addition, many studies have demonstrated that high-dose APAP can induce oxidative stress increases, causing tubular cell death through necrosis or apoptosis pathway . However, there is no evidence that APAP can cause kidney dysfunction by inducing fibroblast proliferation. Here our studies first demonstrate that high-dose APAP (7.94 mM) can inhibit cell survival on kidney tubular cells (NRK-52E) while promoting cell proliferation on kidney interstitial fibroblasts (NRK-49F). Furthermore, 1/10 high-dose (0.794mM) and low-dose (0.0794mM) APAPcan also induce fibroblast proliferation on kidney fibroblasts. Therefore, previous studies and our experimental results indicate that APAP not only inhibits cell survival on kidney tubular cells but also promotes fibroblast proliferation, leading to renal failure.
On the other hand, APAP can induce different cytotoxic mechanisms on different hepatoma cell lines. APAP can induce caspase-dependent apoptosis on hepatoma HUH7 and SK-Hep1 cells , and induce apoptosis and necrosis on hepatoma HepG2 cells . Additionally, a study has demonstrated that high-dose APAP can inhibit DOX-induced cell death on hepatoma HepG2 cells . Although APAP induced-apoptosis on hepatoma Hep3B cells has been observed , the mechanisms of APAP-induced apoptosis on Hep3B cells are still unclear. Our studies further demonstrate that APAP can cause increases in H2O2 levels, and yet cannot induce O2- increases on Hep3B cells. In addition, APAP can activate caspase-9/-3 cascade activation but cannot activate caspase-8/-3 cascade on Hep3B cells. In conclusion, our studies demonstrate that APAP can promote cell proliferation on interstitial fibroblasts and an also induce H2O2 level increases as well as caspase-9/-3 cascades activation, resulting in cell cytotoxic on Hep3B cells.
Materials and methods
Materials. Luminol, Lucigenin, and Hoechst 33342 were obtained from Sigma.
TGF-B was obtained from R&D Systems. MTT assay kit was purchased from
BIO-BASIC CANADA INC. Ac-LEHD-pNA (acetyl-Leu-Glu-His-Asp-p-nitroanilide: caspse-9 substrate), Ac-DEVD-pNA (Acetyl-Asp-Glu-Val-Asp-p-nitroanilide: caspase-3 like substrate), and Ac-IETD-pNA (acetyl-Ile-Glu-Thr-Asp-p-nitroanilide: caspase-8 substrate) were purchased from Anaspec (San Jose, 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
CA). Fetal bovine serum, DMEM, non-essential amino acid, L-glutamine, and penicillin/streptomycin were purchased from GIBCO BRL.
Cell lines and cell cultures. The NRK-52E (rat kidney tubular epithelial cell) and
NRK-49F (rat kidney fibroblast) cell lines were purchased from Bioresource Collection and Research Center (BCRC, Shin Chu, Taiwan). These cell lines were cultured in a DMEM medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin/streptomycin, and 0.1 mM non-essential amino acids and maintained at 37°C in a humidified atmosphere containing 5% CO2 .
Cell survival rate assay. Cell survival rates of NRK-52E, NRK-49F and Hep3B
were determined by using the MTT assay method described in previous study . In brief, cells were cultured in each well of 96-well plates. On the second day, cells were divided into control group and experimental group. MTT assays were determined everyday according to the manufacturer’s instructions. Absorbance at 570 nm was determined under a multi-well ELISA reader (Molecular Devices).
H2O2 and O2- levels determination. H2O2 and O2- levels were measured by lucigenin-amplied method . Briefly, 200 ul of sample was mixed with 0.2 mmol/L of luminol solution (100 ul) then was measured by the chemiluminescence analyzing system (CLA-FSI, Tohoko Electronic Industrial Co. Ltd in Japan) for H2O2 levels determination. 200 ul of sample was mixed with 0.1 mmol/L of lucigenin solution (500 ul) then was measured by the CLA-FSI chemiluminescence analyzing system for O2- levels determination.
Nuclear observation. Nuclear morphology was observed by nuclear staining with
Hoechst 33342. Cells were treated with Hoechst 33342 (10 ug/ml) for 10 min. Nuclear condensation and DNA fragmentation were observed under a fluorescence microscope (excitation: 352 nm; emission: 450 nm) .
Caspase activity assay. Cells were treated with a lysis buffer (50 mM Tris-HCl,
120 mM NaCl, 1 mM EDTA, 1% NP-40, pH 7.5) and then protease inhibitors were added. Cell pellets were obtained through centrifugation (15,000×g, 4C, 30 min). Caspase-3, -8, and -9 activities were determined based on previous studies . 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155
In brief, the working solutions were prepared containing 40 μl cell lysates(80 μg total protein), 158 μl reaction buffer (20% glycerol, 0.5 mM EDTA, 5 mM dithiothreitol, 100 mM HEPES, pH 7.5), and 2 μl fluorogenic substrate (Ac-LEHD-pNA, Ac-DEVD-pNA, or Ac-IETD-pNA). Then the working solutions were incubated at 37C for 6 hours. The level of fluorogenic substrate cleavage is detectable at 405 nm in an ultra-microplate reader (Bio-Tek instruments). Fold increase in caspase activity was calculated using the following formula: (A405sample A405control) / A405control.
Statistical analysis. Data were obtained from four independent triplicate
experiments and are presented as the mean values of the chosen triplicate groups. These data are shown as means with standard deviations.
Results
Acetaminophen (APAP) decreases survival rate on kidney tubular epithelial cells while inducing proliferation on kidney fibroblasts. Similar to previous studies,
high-dose APAP (> 5 mM) can cause cell cytotoxicity on cell models . Our study also showed that high-dose APAP (7.94 mM) can decrease cell survival rate on kidney tubular epithelial cells (NRK-52E cells) in a time-dependent manner (Figure 1A). Compared with high-dose APAP-treated NRK-52E cells, survival rate did not decrease obviously on NRK-52E cells with 1/10 high-dose APAP treatment (Figure 1A). These results suggest that APAP-induced cell cytotoxicity is dependent on APAP concentration and incubation time. However, to our surprise, although high-dose APAP decreases survival rate on NRK-52E cells, high-dose APAP is able to promote cell proliferation on kidney fibroblasts (NRK-49F cells) (Figure 1B). On the other hand, 1/10 high-dose APAP can also induce cell proliferation on NRK-49F cells (Figure 1B). It is well-known in clinical cases that both tubular epithelial cell damage and kidney fibrosis can induce renal failure . Therefore, our findings indicate the possibility that APAP-induced renal failure may not only inhibit tubular epithelial cell survival but also promote renal fibroblast proliferation.
Low-dose APAP induces cell proliferation on kidney fibroblasts. Previous studies
have demonstrated that high-dose APAP can inhibit tubular epithelial cells 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187
survival to induce renal failure . Here, as shown in Figure 1, high-dose APAP can cause growth inhibition on tubular epithelial cells, and our study demonstrate that APAP can induce cell proliferation on kidney fibroblasts. For kidney fibrosis patients, it is important to prevent fibroblast proliferation from worsening their condition. In order to provide more helpful information about APAP treatment for fibrosis patients, it is valuable to understand if low-dose APAP (below therapeutic dose) can induce fibroblast proliferation. Therefore, in this study, dose APAP is applied to kidney fibroblasts to further investigate how low-dose APAP affects cell growth of kidney fibroblasts. Looking at our results, it is worth noticing that low-dose APAP cannot inhibit cell survival on NRK-52E cells while low-dose APAP can induce cell proliferation on NRK-49F cells (fibroblast) (Figure 2A). In addition, APAP can induce fibroblast proliferation similar to TGF-B-treated fibroblast in a dose-dependent manner (Figure 2B). As is well known, below the therapeutic dose, APAP has not been toxic to liver and kidney cells in clinical cases. However, this APAP dose is able to induce fibroblast proliferation, which may harm fibrosis patients. Thus, our study provides useful information suggesting that fibrosis patients must pay attention to APAP treatment.
APAP exerts more significant cell cytotoxic effects on Hep3B cells than on NRK52 cells. Compared with APAP-treated NRK-52E cells (tubular cells),
high-dose APAP also induces cell cytotoxic effect on Hep3B cells (hepatoma cells) (Figure3A). The data shows that cell survival rate on high-dose APAP-treated Hep3B cells is lower than APAP-treated NRK-52E cells. On the other hand, at 1/10 high-dose concentration, APAP does not cause obvious cytotoxic effects on NRK-52E cells, but it can induce cytotoxic effects on Hep3B cells (Figure 3B). Therefore, APAP exerts more significant cytotoxic effects on Hep3B cells than on NRK-52E cells. These results indicate that APAP, at a non-toxic concentration to normal tubular cells, may exert an anti-tumor activity on hepatoma cells.
APAP induces H2O2 level increases and apoptosis on Hep3B cells. APAP
induced-cell cytotoxic effects related to Reactive Oxygen Species (ROS) increase have been demonstrated . However, it is still unclear which ROS can be 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219
increased with APAP treatment. Both O2- and H2O2 are generallt found in cells. O2- and H2O2 levels are determined in this study, and it is found that APAP can induce increases in H2O2 levels (Figure 4A) but cannot induce O2- level increases in Hep3B cells (Figure 4B). Therefore, APAP-induced cell cytotoxicity is possibly related to H2O2 but not related to O2-. On the other hand, observations on nuclear morphology, nuclear condensation, and DNA fragmentation are found on APAP-treated Hep3B cells (Figure 5). The results suggest that APAP can induce cell cytotoxicity via H2O2 level increases.
APAP activates caspase-9/-3 cascade on Hep3B cells. Caspase activation can
induce cell apoptosis . Two major caspase cascades have been reported including caspse-9/-3 pathway and caspse-8/-3 pathway. APAP can induce apoptosis on Hep3B as shown in Figure 5. Therefore, caspase activities are determined in this study. Using substrate cleavage assay , caspase-9 and caspse-3 activities are found on APAP-treated Hep3B cells (Figure 6A and 6C). However, caspase-8 activity is not found obviously on Hep3B cells (Figure 6B). Therefore, our study suggests that APAP can activate caspase-9/-3 cascades to induce cell cytotoxicity on Hep3B cells.
Discussion
Both tubular epithelial cell damage and fibroblast proliferation can induce renal dysfunction . Many studies have demonstrated that APAP overdose can inhibit tubular epithelial cells survival, resulting in nephrotoxicity . Most of these studies focused on high-dose APAP-induced acute intoxication on kidney tubular cells. Therefore, patients should obtain more APAP information and take notice of APAP treatment to prevent APAP-induced acute damage. However, it is still unclear whether low-dose APAP will likely cause chronic kidney damage. Our present study demonstrates that not only can high-dose APAP decrease cell survival on tubular epithelial cells, but it can also induce proliferation on fibroblasts even with low-dose treatments. That is, APAP induced-renal damage may occur through epithelial cell damage or fibroblast proliferation. In general, acute damage is easier to detect and diagnose than chronic damage, therefore, APAP overdose-induced acute intoxication is commonly noted, whereas low-dose APAP-induced damage may be ignored in clinical cases. Here we 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252
demonstrate that low-dose APAP can promote fibroblast proliferation. Thus we consider therapeutic dose APAP to be a safe analgesic and antipyretic agent for non-fibrosis patients, but it may be harmful to kidney fibrosis patients.
TGF-B signal pathways are closely related to renal damage . TGF-B induced-renal damage is correlated with: 1) TGF-B-induced tubular cell death ; 2) TGF-B-induced epithelial mesenchymal transition ; 3) TGF-B-induced fibroblast proliferation . Up to now, no evidence has shown that APAP can induce kidney fibroblast proliferation via TGF-B-related signals. Compared with TGF-B-treated kidney cells, APAP also inhibits tubular cell survival and promotes fibroblast proliferation in this study. In addition, a previous study showed that TGF-B were significantly elevated in APAP-treated liver tissue [68]. Based on these resaons, we suggest that APAP inhibits kidney tubular cell survival and induces kidney fibroblast proliferation corelated with TGF-B signal pathway. However, whether or not APAP has epithelial mesenchymal transition effects on kidney tubular cells, like it does on TGF-B-treated tubular cells, should be studied in the future.
O2- and H2O2 belong to ROS and are usually found in cells. They are generated normally in the electric transport chain. O2- can be cleaned by superoxide dismutase and H2O2 can be cleaned by catalase or glutathione system. It is well known that cells will be damaged when O2- and H2O2 levels are raised. Previous studies have demonstrated that APAP overdose can induce an increase of ROS levels to cause decreases in cell viability . However, these studies did not directly demonstrate which ROS is raised under APAP treatment. Here, two types of ROS (O2- and H2O2) are determined. H2O2 levels increase, but there is no significant change to O2- levels on APAP-treated cells. Our study suggests that the inhibiting of cell survival by APAP may occur through H2O2 level increases at the early stage. This is a possible reason for why N-acetyl cysteine (NAC), a material for glutathione synthesis, is applied for clinical APAP-induced poisoning in emergency cases .
APAP-induced cell death has been studied widely . These studies demonstrate that APAP induces cell death either via apoptosis or necrosis death pathway on different cells. In our present study, apoptosis characteristics are observed on APAP-treated Hep3B cells, similar to previous studies [48, 72]. Moreover, our study further demonstrates that caspase-9/-3 cascade is activated 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286
while caspase-8/-3 cascade is not activated. Caspase-9/-3 cascade-related to mitochondrial damage and caspase-8/-3 cascade-related to death receptor signals have been reported previously . Thus, our data suggest that APAP-induced cell cytotoxicity correlates with mitochondrial damage on Hep3B cells. Finally, previous studies have shown APAP-induced cytotoxicity under high-dose APAP (> 5mM) treatment on cell models . In this study, high-dose APAP can induce cytotoxicity on normal kidney tubular cells and hepatoma cells. However, 1/10 high-dose APAP is only cytotoxic to hepatoma cells. That is, non-toxic dose APAP (to normal cells) may be considered as an anti-tumor drug in the future.
Overall, this study shows that: 1) APAP can induce cell proliferation on kidney fibroblasts even under low-dose treatments, and thus we suggest that fibrosis patients must be careful with APAP treatment; 2) APAP can induce H2O2 level increases and activate caspase-9/-3 cascade to cause cell cytotoxicity; 3) APAP induces different cytotoxicity on different cell types, and our study shows that cell cytotoxicity is observed more significantly on APAP-treated hepatoma cells than on APAP-treated normal kidney tubular cells.
Acknowledgments
This work was supported by the following grants: NSC99-2320-B-039-030-MY3; NSC99-2632-B-039-001-NSC99-2320-B-039-030-MY3; NSC101-2321-B-039-004; and NHRI-EX102-10245BI.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure Legends:
Figure 1. Effects of high-dose APAP on kidney cell survival rates. (A) NRK-52E cells were treated with high-dose and 1/10 high dose APAP. (B) NRK-49F were treated with high-dose and 1/10 high dose APAP. Survival rates of cells were calculated daily using the MTT assay method. Data was analyzed from four independent experiments and presented as mean S.D.
Figure 2. Effects of low-dose APAP on kidney cell survival rates. (A) NRK-52E cells and NRK-49F cells were treated with low-dose APAP. (B) NRK-49F cells were treated with high-dose, 1/10 high dose, low-dose APAP, and TGF-B. TBF-B-treated group was a positive control. Survival rates of cells were calculated daily using the MTT assay method. Data was analyzed from four independent experiments and presented as mean S.D.
Figure 3. Effects of APAP on hepatoma cell survival rates. (A) NRK-52E cells and Hep3B cells were treated with high-dose APAP. (B) NRK-52E cells and Hep3B cells were treated with 1/10 high-dose APAP. Survival rates of cells were calculated daily using the MTT assay method. Data was analyzed from four independent experiments and presented as mean S.D.
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Figure 4. Effects of APAP on O2- and H2O2 levels. (A) O2- levels were determined on control cells, high-dose-treated and 1/10 high-dose APAP-treated cells. (B) O2- levels were determined on control cells, high-dose-APAP-treated, and 1/10 high-dose APAP-treated cells. O2- and H2O2 levels were determined after APAP treatment for 6 hours using a lucigenin-amplied method . Data was analyzed from four independent experiments and presented as mean S.D.
Figure 5. Effects of APAP on nuclear condensation and DNA fragmentation. (A) Control cells (B) APAP-treated cells. After cells were treated with APAP for 72 hours, nuclear morphology was observed by nuclear staining with Hoechst 33342 treatment. Note that nuclear condensation (yellow arrow) and DNA fragmentation (white arrow) were found on APAP-treated cells.
Figure 6. Caspases activity. Caspase-9 (A) caspase-8 activation (B) and caspase-3 activities were determined on control cells and APAP-treated cells. Note that caspase-3 and caspase-9 activities significantly increased in APAP-treated cells. Data was obtained from three independent experiments and presented as mean S.D.
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