An extract of Rhodobacter sphaeroides reduces cisplatin-induced nephrotoxicity in
mice
Wen-Wei Chang 1,2, Jau-Jin Liu 3, Chi-Fan Liu 3, Wen-Sheng Liu 4, Yun-Ping Lim
5, Yu-Jung Cheng 6 and Che-Hsin Lee 3,*
1
Department of Biomedical Sciences, College of Medical Science and
Technology, Chung Shan
Medical University, Taichung 402, Taiwan. Email: changww@csmu.edu.tw
2
Department of Medical Research, Chung Shan Medical University Hospital,
Taichung 402, Taiwan
3
Department of Microbiology, School of Medicine, China Medical University,
Taichung 404,
Taiwan. Email: JJL: jjl@mail.cmu.edu.tw; CFL:
ruz0417@yahoo.com.tw;CHL:chlee.cmu.edu.tw
4
Asia-Pacific Biotech Developing, Inc. Kaohsiung, 806, Taiwan. Email:
wensheng5394@gmail.com
5
Department of Pharmacy, College of Pharmacy, China Medical University,
Taichung 404, Taiwan. Email:limyp@mail.cmu.edu.tw
6
Department of Physical Therapy, Graduate Institute of Rehabilitation
Science, China Medical University, Taichung 404, Taiwan. Email:
chengyu@mail.cmu.edu.tw
*
Corresponding author: Che-Hsin Lee, Department of Microbiology, School of
Medicine, China Medical University, Taichung 404, Taiwan; E-Mail:
chlee@mail.cmu.edu.tw
Tel.: +886-4-22053366-2173; Fax: +886-4-22053764.
Received: / Accepted: / Published:
Abstract: Cisplatin is used as a treatment for various types of solid tumors. Renal
injury severely limits the use of cisplatin. Renal cell apoptosis, oxidative stress, and
inflammation contribute to cisplatin-induced nephrotoxicity. Previously, we found
that an extract of Rhodobacter sphaeroides (LycogenTM) inhibited proinflammatory
cytokines and the production of nitric oxide in activated macrophages in a dextran
sodium sulfate (DSS)-induced colitis model. Here, we evaluated the effect of
Lycogen™, a potent anti-inflammatory agent, in mice with cisplatin-induced renal
injury. We found that attenuated renal injury correlated with decreased apoptosis due
to a reduction in caspase-3 expression in renal cells. Oral administration of
Lycogen™ significantly reduced the expression of tumor necrosis factor-α and
interleukin-1β in mice with renal injury. LycogenTM reduces renal dysfunction in
mice with cisplatin-induced renal injury. The protective effects of the treatment
included blockage of the cisplatin-induced elevation in serum urea nitrogen and
creatinine. Meanwhile, Lycogen™ attenuated body weight loss and significantly
prolonged the survival of mice with renal injury. We propose that Lycogen™ exerts
anti-inflammatory activities that represent a promising strategy for the treatment of
cisplatin-induced renal injury.
Keywords: Rhodobacter sphaeroides; Lycogen™; cisplatin; nephrotoxicity
ABSTRACT
Bacteria can produce some compounds in response to their environment. These
compounds are widely used in cosmetic and pharmaceutical applications. Some
probiotics have immunomodulatory activities and modulate the symptoms of several
diseases. Renal injury severely limits the use of cisplatin. Renal cell apoptosis,
oxidative stress, and inflammation contribute to cisplatin-induced nephrotoxicity.
Previously, we found that the extracts of Rhodobacter sphaeroides (LycogenTM)
inhibited nitric oxide production and inducible nitric-oxide synthase expression in
activated macrophages. Meanwhile, the effect of LycogenTM, a potent
anti-inflammatory agent, was evaluated in mice with dextran sodium sulfate
(DSS)-induced colitis. In this study, the effect of Lycogen™, a potent
anti-inflammatory agent, was evaluated in mice with cisplatin-induced renal injury.
We demonstrated that the attenuated renal injury correlated with decreased apoptosis
that is associated with reduction in caspase-3 expression in renal cells. Oral
administration of Lycogen™ reduced the expressions of proinflammatory cytokines
(tumor necrosis factor-α and interleukin-1β) in mice after cisplatin treatment. In
addition, the decreased expression of blood urea nitrogen and creatinin in the serum
induced by cisplatin was ameliorated by Lycogen™. Meanwhile, Lycogen™ reduced
the loss of body weight and dramatically prolonged the survival of mice with renal
injury. These findings identified that Lycogen™ is an anti-inflammatory agent with the
capacity to ameliorate cisplatin-induced renal injury.
Keywords: Rhodobacter sphaeroides; Lycogen™; cisplatin; nephrotoxicity
INTRODUCTION
Cisplatin, a platinum anticancer drug, has been wildly used for the treatment of
malignant tumors. The important dose-limiting factors of cisplatin in nephrotoxicity.
Renal cell apoptosis, oxidative stress, and inflammation have been recognized as the
mechanism of cisplatin-induced nephrotoxicity (Pabla and Dong 2008).
Cisplatin-induced cell death is the major mechanism in renal tubule cells.
Some nutritional and natural components have been studied for their effectiveness in
preventing cisplatin-induced renal injury by controlling production of inflammatory
cytokines (Wu et al., 2008; Hsiang et al., 2013). Furthermore, microorganisms such as
fungi and bacteria produce many metabolites that have been developed into drugs.
Bacteriopurpurinimides were derived from the phototrophic bacteria, Rhodobacter
sphaeroides (Chen et al.,2002). Carotenoids are naturally occurring compounds also
found in R. sphaeroides (Deshmukh et al., 2011). Studies found that carotenoids
having anti-oxidant activity are able to inhibit tumor growth (Kelsey et al.,
2012).Therapeutic strategies utilizing nature products may enhance host immune
balance and decrease renal injury after cisplatin treatment. Previous our study has
demonstrated that the extract of R. sphaeroides (Lycogen™) display
anti-inflammatory, and anti-oxidative activities that provide organ-protective potential
(Liu et al., 2012). In this study, we identified a novel strategy by exploiting the
Lycogen™, aiming at preventing murine cisplatin-induced renal injury using its
anti-inflammatory effects.
MATERIAL AND METHODS
Reagents, cells and mice
R. sphaeroides (WL-APD911) was isolated from mutants using chemical mutagenesis
[Bioresource Collection and Research (BCRC), Hsinchu, Taiwan]. The R.
sphaeroides was cultured in broth. After harvesting, the bacterial broth was
centrifuged and washed with ethanol. The bacterial residue is extracted with acetone
and then centrifuged by 7,500 rpm for 5 min. The supernatant is filtered through filter
paper and a 0.2 μm filter into a round-bottomed flask. The color of the final
supernatant is dark red. Acetone is removed completely in oven at 55 °C. The R.
sphaeroides extract was named Lycogen™ (Liu et al., 2012). Lycogen™ was
dissolved in PBS. The MES-13 cell line (glomerular mesangial cells from an SV40
transgenic mouse) was obtained from American Type Culture Collection (CRL-1927;
Manassas, VA) and maintained in culture medium with a 3:1 mixture of DMEM and
Ham's medium, supplemented with 14 mM HEPES, 2 mM glutamine, antibiotics
(100 μg/ml penicillin and 100 μg/ml streptomycin) and 5% FBS at 37 °C. The
incubation chamber was equilibrated with 5% CO2–95% air. Male C57BL/6 mice
were obtained from the Taiwan National Laboratory Animal Center. The
experimental protocol adhered to the rules of the Animal Protection Act of Taiwan.
The experimental protocol has been approved by the Laboratory Animal Care and Use
Committee. All mice were kept under standard conditions of temperature and light, and
were fed with standard laboratory chow and water. Cisplatin was purchased from
Sigma-Aldrich (St. Louis, MO, USA).
Assay of cell proliferation
Cells (105/well) were treated with various concentration of LycogenTM or cisplatin
in culture medium. The medium were removed, washed, and replenished with fresh
medium supplemented with 2% FBS. Cell proliferation was assessed by the
colorimetric WST-1 assay (Dojindo Labs, Tokyo, Japan) according to the
manufacturer’s instructions (Lee et al., 2013).
Western blot analysis
Cell lysates were prepared by extracting proteins with lysis buffer. Proteins from total
cell extracts were fractionated on SDS-PAGE, transferred onto Hybond enhanced
chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK), and
probed with primary antibodies against caspase 3 (GeneTeX, San antonio, TX, USA)
or monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich). Horseradish
peroxidase-conjugated secondary antibodies were used, and protein-antibody
complexes were visualized by enhanced chemiluminescence system (Amersham)
(Chen et al., 2012).
Establishment of experimental renal injury model
Male C57BL/6 mice at 6–8 weeks of age were intraperitoneal (i.p.) cisplatin (30mg/ml)
as previously described (Mitazaki et al., 2013).To study the therapeutic effect of
Lycogen™, this substance (1 mg/kg/day) was orally administered for three
consecutive days by gavage. Control mice were treated with PBS. Mice were i.p.
injected cisplatin at day 3. Body weight loss is calculated as the percent difference
between the original body weight and the actual body weight daily
Determination of creatinine and BUN
The groups of mice were treated with Lycogen™ (1 mg/kg) by oral administration
daily for three consecutive days by gavage and then mice were i.p. injected cisplatin
(30mg/kg) at day 3Renal function was assessed by determination of creatinine and
BUN using creatinine assay kit (BioVision, Milpitas, CA, USA) and urea assay kit
(BioVision) at day 6.
Assessment of Cytokines
To determine the expression of TNF-α and IL-1β, the groups of mice were treated
with Lycogen™ (1 mg/kg) by oral administration daily for three consecutive days by
gavage and then mice were i.p. injected cisplatin (30mg/kg) at day 3. To detect
cytokine expressions, the sera were collected at day 6. Levels of cytokines in the sera
were determined by an enzyme-linked immunosorbent assay (ELISA, R＆D,
Minneapolis, MN, USA) (Lee et al., 2011a; Lee et al., 2011b).
Statistical Analysis
All data were expressed as mean ± standard deviation (SD). The unpaired,
two-tailed Student’s t
test was used to determine differences between groups. The mice survival analysis
was performed using the Kaplan-Meier survival curve and log-rank test. Any P value
less than 0.05 is considered statistically significant.
RESULTS
Evaluation of cell viability after LycogenTM or cisplatin treatment in vitro
In this study, we used mouse glomerular mesangial cell (MES-13) to evaluate the
potential cytotoxic effect of LycogenTM and cisplatin. The results for in vitro treatment
of MES-13 cells with LycogenTM for cell survival, and melanin content are shown in
Figure 1. Thus dose (2 μM-16μM) without significant cytotoxicity were observed (Fig.
1A). The cell viability of MES-13 cells dramatically decreased after treatment with
cisplatin (Fig. 1B). Taken together, these results suggest that LycogenTM showed no
cytotoxic effects on MES-13 cell compared with the cells treated with cisplatin.
LycogenTM treatment attenuated cisplatin-induced renal cell death
We have evaluated the effect of LycogenTM and cisplatin in MES-13 cells and found
that MES-13 cells pretreated with LycogenTM significantly reduced the cell death
induced by cisplatin (Figure 1A). Meanwhile, as shown in Fig. 2B, LycogenTM
dose-dependently reduced the expression of caspase 3 in MES-13 cells. Our results
clearly demonstrate that LycogenTM treatment markedly attenuated the renal cell death
after cisplatin treatment.
LycogenTM treatment attenuated renal injury is cisplatin-treated mice
Cisplatin administration caused 2- and 3-fold increase in creatinine and blood urea
nitrogen (BUN). Treatment with LycogenTM led to 27% reduction in creatinine and
38% BUN. No significant differences were observed in creatinine and BUN between
mice treated with PBS or LycogenTM (Figu. 3). To evaluate the effect of LycogenTM
treatment on cisplatin-induced renal inflammation, we measured the protein
expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β).
As shown in Fig. 4, cisplatin induced protein expression of proinflammatory
cytokines, such as IL-1β and TNF-α, in sera, whereas Lycogen™ suppressed the host
protein expressions of proinflammatory cytokines. These results pointed out that oral
administration of Lycogen™ inhibited the production of proinflammatory cytokine in
the cisplatin-induced renal inflammation model.
Lycogen™ Improved cisplatin-Induced body weight loss and survival
We further monitored symptomatic renal failure parameters including body weight
and survival caused by renal injury 3 days after starting cisplatin (30mg/kg)
administration (Fig. 2). Treatment of mice with Lycogen™ significantly attenuated
the weight loss induced by cisplatin (Fig. 5A). Oral Lycogen™ did not influence the
body weight and survival of mice, suggesting that Lycogen™ was safe for mice.
Furthermore, Lycogen™ dramatically prolonged the survival of mice with severe
renal injury (Fig.5B).
DISCUSSION
Cisplatin is one of the most used chemotherapeutic drugs. Meanwhile, the major
limiting factor in the use of cisplatin is the side effects in normal tissue including
kidney. Renoprotective approaches are being discovered, but the effects are partial.
The current study demonstrated that a bacteria-derived molecule could improve the
epithelial cell injury (Okamoto et al., 2012). Mice fed diets containing Lycogen™ (1
mg/kg) for 3 days showed no effect on body weight, and proinflammatory cytokine
productions. Moreover, no adverse side effects were observed when the diet contained
Lycogen™ (Fig. 5B). Oxidative stress was observed in patients with nephrotoxicity
(Sahu et al., 2013). These findings implied that the anti-oxidative potential of
Lycogen™ have the activity to inhibit the cisplatin-induced renal injury. Inflammatory
cytokines play important role in the pathogenesis of nephrotoxicity (Sahu et al., 2013).
High levels of the pro-inflammatory cytokines, IL-1β and TNF-α, are present in
patients suffering from nephrotoxicity. There is growing recognition the importance of
inflammation in the pathogenesis of cisplatin-induced nephrotoxicity. Cisplatin
administration caused 4- and 8-fold increase in TNF-α and IL-1β. Treatment with
LycogenTM led to 42% reduction in TNF-α and 35% IL-1β. Lycogen™ contains
ζ-carotene, neurosporene, spheroidenone and methoxyneurosporene according to
nuclear magnetic resonance spectroscopy analysis. ζ-Carotene is the precursor of
neurosporene, which in turn is the precursor of lycopene (Albrecht et al., 1995).
Lycopene, as an anti-inflammatory agent, prevents the production of inflammatory
cytokines (Ghavipour et al., 2012). Meanwhile, neurosporene itself has the ability to
protect irradiation with UV-B (Sandmann et al., 1998). Further work is warranted to
elucidate the active ingredient(s) in Lycogen™. These findings point out that the
anti-oxidative and anti-inflammatory potential of Lycogen™ might contribute to its
therapeutic effect on cisplatin-induced nephrotoxicity. In conclusion, our work has
identified Lycogen™ as an anti-inflammatory agent with the capacity to ameliorate
cisplatin-induced nephrotoxicity. However, further work is warranted to elucidate the
underlying mechanism of the therapeutic effects of Lycogen™ therapy in the
cisplatin-induced nephrotoxicity.
Contract grant sponsor: National Science Council, Taiwan
Contract grant number: NSC 101-2320-B-039-012-MY3
FIGURE LEGENDS
Fig. 1. Effects of LycogenTM and cisplatin on cell viability in MES-13 cell.
MES-13 cells were treated with indicated concentrations of LycogenTM and ciplatin for
48 h. Cell viability was measured after (A) LycogenTM treatment or (B) cisplatin
treatment by WST-1 assay.
＊＊＊
, P＜0.001. (mean ± SD, n = 6). Each experiment was
repeated three times with similar results.
Fig. 2. LycogenTM reduced the cisplatin-induced cell apoptosis. MES-13 cells were
pretreated with LycogenTM at the concentration of 0, 2, 4, 8 or 16 μM for 48 h and
then cells were added cisplatin (5μg/ml) for 48 h. (A) Cell viability was measured by
WST-1 assay. (B) The expression of caspase 3 was measure by Western blot analyses.
＊
＊＊
, P＜0.05
, P＜0.01;
＊＊＊
, P＜0.001. (mean ± SD, n = 6). Each experiment was
repeated three times with similar results.
Fig. 3. LycogenTM ameliorated cisplatin-induced renal dysfunction. Effect of
LycogenTM on (A) creatinine and (B) blood urine nitrogen (BUN) levels after cisplatin
administration.
＊
, p＜0.05. (mean ± SD, n = 4). Each experiment was repeated three
times with similar results.
Fig. 4. LycogenTM ameliorated cisplatin-induced renal inflammation. Effect of
LycogenTM on (A) TNF-α and (B) IL-1β levels after cisplatin administration.
＊
, P＜
0.05. (mean ± SD, n = 4). Each experiment was repeated three times with similar
results.
Fig. 5. Effect of Lycogen™ on cisplatin-induced renal injury mice. (A) The mice
with colitis were orally administered with Lycogen™ (1 mg/kg) for three consecutive
days after cisplatin (30mg/kg) induction and the body weights of mice were
determined (means ± SD, n = 6). P < 0.01 for cisplatin-induced renal injury mice
pretreated with Lycogen™ versus cisplatin-induced renal injury mice pretreated with
PBS (B) Kaplan-Meier survival curves on day 14 are shown. (mean ± SD, n = 6)
Significance of differences between groups for continuous variables was assessed using
the Student t-test. The mice survival analysis was performed using the Kaplan-Meier
survival curve and log-rank test. * p < 0.05.
REFERENCES
Albrecht M, Klein A, Hugueney P, Sandmann G, Kuntz M. 1995. Molecular
cloning and functional expression in E. coli of a novel plant enzyme mediating
zeta-carotene desaturation. FEBS Lett 372: 199–202.
Chen MC, Chang WW, Kuan YD, Lin ST, Hsu HC, Lee CH. 2012. Resveratrol
inhibits LPS-induced epithelial-mesenchymal transition in mouse melanoma
model. Innate Immun 18: 685-693.
Chen Y, Graham A, Potter W, Morgan J, Vaughan L, Bellnier DA. Henderson BW,
Oseroff A, Dougherty TJ, Pandey RK.2002. Bacteriopurpurinimides: Highly
stable and potent photosensitizers for photodynamic therapy. J Med Chem 45:
255–258.
Deshmukh SS, Tang K, Kálmán L. 2011. Lipid binding to the carotenoid binding
site in photosynthetic reaction centers. J Am Chem Soc 133: 16309–16316.
Ghavipour M, Saedisomeolia A, Djalali M, Sotoudeh G, Eshraghyan MR,
Malekshahi MA, Wood LG. 2012. Tomato juice consumption reduces systemic
inflammation in overweight and obese females. Br J Nutr 15: 1–5.
Hsiang CY, Lo HY, Huang HC, Li CC, Wu SL, Ho TY. 2013. Ginger extract and
zingerone ameliorated trinitrobenzene sulphonic acid-induced colitis in mice via
modulation of nuclear factor-κB activity and interleukin-1β signaling pathway.
Food Chem 136: 170–177.
Kelsey L, Katoch P, Johnson KE, Batra SK, Mehta PP. 2012. Retinoids regulate
the formation and degradation of gap junctions in androgen-responsive human
prostate cancer cells. PLoS One 7: e32846.
Lee CH, Hsieh JL, Wu CL, Hsu HC, Shiau AL. 2011a. B cells are required for
tumor-targeting Salmonella in host. Appl Microbiol Biotechnol 92: 1251-1260.
Lee CH, Hsieh JL, Wu CL, Hsu PY, Shiau AL. 2011b. T cell augments the
antitumor activity of tumor-targeting Salmonella. Appl Microbiol Biotechnol 90:
1381-1388.
Lee CH, Lin YH, Hsieh JL, Chen MC, Kuo WL. 2013. A polymer coating applied
to Salmonella prevents the binding of Salmonella-specific antibodies. Int J Cancer
132: 717-725.
Liu WS, Chen MC, Chiu KH, Wen ZH, Lee CH. 2012. Amelioration of dextran
sodium sulfate-induced colitis in mice by Rhodobacter sphaeroides extract.
Molecules 17: 13622-13630.
Mitazaki S, Hashimoto M, Matsuhashi Y, Honma S, Suto M, Kato N,
Nakagawasai O, Tan-No K, Hiraiwa K, Yoshida M, Abe S. 2013. Interleukin-6
modulates oxidative stress produced during the development of cisplatin
nephrotoxicity. Life Sci 92: 694-700.
Okamoto K, Fujiya M, Nata T, Ueno N, Inaba Y, Ishikawa C, Ito T, Moriichi K,
Tanabe H, Mizukami Y, et al. 2012. Competence and sporulation factor derived
from Bacillus subtilis improves epithelial cell injury in intestinal inflammation via
immunomodulation and cytoprotection. Int J Colorectal Dis
27: 1039–1046.
Pabla N, Dong Z. 2008. Cisplatin neophrotoxicity: Mechanisms and
renoprotective strategies. Kindy Int 73: 994-1007.
Sahu BD, Kuncha M, Sindura GJ, Sistla R. 2013. Hesperidin attenuates
cisplatin-induced acute renal injury by decreasing oxidative stress, inflammation
and DNA damage. Phytomedicine 20: 453-460.
Sandmann G, Kuhn S, Böger P. 1998. Evaluation of structurally different
carotenoids in Escherichia coli transformants as protectants against UV-B
radiation. Appl Environ Microbiol 64: 1972–1974.
Wu SL, Chen JC, Li CC, Lo HY, Ho TY. Hsiang CY. 2009. Vanillin improves and
prevents trinitrobenzene sulfonic acid-induced colitis in mice. J Pharmacol Exp
Ther 330: 370–376.