Combined treatment of carfilzomib and z-VAD-fmk inhibits skeletal proteolysis and apoptosis and ameliorates cancer cachexia

Page 1

ORIGINAL PAPER

Combined treatment of carfilzomib and z-VAD-fmk inhibitsskeletal proteolysis and apoptosis and ameliorates cancer cachexia

Qiang Wang • Chunhong Li • Xudong Peng • Qingjie Kang • Dawei Deng •

Liuping Zhang • Yueyong Zheng • Chaoyi Wang • Zhongpeng Qiao •

Dunwei Guo • Song You • Hua Tang

Received: 3 January 2015 / Accepted: 13 February 2015

� Springer Science+Business Media New York 2015

Abstract The purpose of the study was to evaluate the

therapeutic benefit of treatments with carfilzomib (CFZ)

and z-VAD-fmk in a mouse model of cancer-induced

cachexia. The model of cancer-associated cachexia was

generated by injecting murine C26 adenocarcinoma cells

into BALB/C mice. CFZ and z-VAD-fmk were adminis-

tered individually or in combination at 5 and 12 days after

inoculation. Changes in body weight, gastrocnemius mus-

cle mass, tumor burden, spontaneous activity, survival, and

metabolic profiles were noted. Also evaluated were the

circulatory levels of renin and angiotensin II, and levels of

apoptotic, proteolytic, and renin-angiotensin system-asso-

ciated markers and transcription factor 2 (ATF2) in gas-

trocnemius muscle. The CFZ and z-VAD-fmk treatments

were associated with less muscle wasting, reduced tumor

burden, modulated metabolism, higher levels of glucose,

albumin, and total proteins, and lower levels of triglyceride

fatty acids, more spontaneous physical activity, and longer

survival in C26-inoculated mice compared with PBS-

treated cachectic mice. CFZ and z-VAD-fmk treatments

resulted in higher levels of caspase-3 and BAX and lower

level of BCL-XL in gastrocnemius muscles and altered the

level of proteins in the renin-angiotensin system. The

combined treatment administered 5 days after C26

inoculation was more effective than other regimens.

Combined treatment with CFZ and z-VAD-fmk early in the

development of cachexia was associated with signs of less

proteolysis and apoptosis and less severe cachexia in a

mouse model of cancer-induced cachexia.

Keywords Carfilzomib � z-VAD-fmk � pATF2 �Proteolysis � Apoptosis

Introduction

Cancer cachexia is a multifactorial syndrome characterized

by ongoing loss of skeletal muscle mass that is not fully

responsive to conventional treatments such as nutritional

support [1]. In cancer patients, especially patients with

advanced disease, the incidence of cachexia is high, rang-

ing from 50 to 80 % [2, 3]. Cachexia not only reduces the

quality of life of cancer patients and their caregivers, but

also obstructs patients’ response to treatment and reduces

their ability to tolerate treatment-associated toxicity [3–5].

Over 30 % of cancer-related deaths can be attributed to

cachexia [2]. To identify potential therapies for cachexia

and develop effective treatments, it is important to obtain a

better understanding of the underlying mechanism of this

detrimental syndrome.

The most prominent feature of cachexia is skeletal

muscle wasting, a process during which excessive prote-

olysis of myofibrils occurs. The role of the ubiquitin–pro-

teasome system (UPS) in skeletal muscle proteolysis has

been well established [6, 7]. In addition, the presence of

DNA fragments in skeletal muscle during muscle wasting

suggests that an apoptotic pathway is activated in this

catabolic state.

Caspase-3, one of the key players in apoptosis, was

activated at the initial step to break down the myofibril,

providing protein substrates for UPS [8–10]. Caspase-3

Q. Wang � C. Li � X. Peng � Q. Kang � D. Deng � L. Zhang �Y. Zheng � C. Wang � Z. Qiao � D. Guo � S. You � H. Tang (&)

Department of General Surgery, The First Affiliated Hospital of

Chongqing Medical University, YouYi Road, Yuzhong District,

Chongqing 400016, China

e-mail: [email protected]

123

Med Oncol (2015) 32:100

DOI 10.1007/s12032-015-0538-6

Page 2

also cleaves specific 19S proteasome subunits in skeletal

muscle to stimulate proteasome activity [11]. This suggests

that both the UPS and apoptotic pathways are important in

mediating the wasting of skeletal muscle in cachexia.

Interestingly, angiotensin II (ANGII), in the renin-an-

giotensin system (RAS), can activate the UPS and several

proteins in the proteolytic pathway, including caspase 3,

MuRFl, and MAFbx [12]. ANGII also regulates the activity

of activating transcription factor 2 (ATF2), a transcription

factor for renin [13]. Murphy et al. [14] found that ANGII

can induce the expression of MuRFl and MAFbx; an in-

hibitor of the RAS hormone system, specifically an in-

hibitor of angiotensin converting enzyme (ACE), which

mediates conversion of AngI to AngII, enhanced skeletal

muscle function in mice with mild or severe cachexia.

Their results support the contribution of the RAS in

cachexia.

Given the significant contribution of UPS and caspase-3

in skeletal muscle wasting, we hypothesized that inhibiting

UPS and caspase-3 activities may confer therapeutic ben-

efits to patients with cancer-associated cachexia. CFZ is an

epoxyketone-based irreversible 20S proteasome inhibitor

[14] that selectively inhibits the chymotrypsin-like activity

of the proteasome and induces cell death in multiple types

of cancers [15, 16]. Currently, CFZ is approved as a sec-

ond-line treatment for patients with multiple myeloma

[17]. z-VAD-fmk is a widely used broad-spectrum caspase

inhibitor that can protect muscle from compression-in-

duced damage and restores muscle function [18] and im-

pairs angiogenesis, which may inhibit tumor growth [19].

In the current study, we tested various treatments with

Carfilzomib (CFZ) and z-VAD-fmk at different stage of

cachexia in a mouse model of cancer-associated cachexia.

To further understand the molecular mechanism underlying

the therapeutic benefit of these treatments, we also char-

acterized the expression of various apoptotic, proteolytic,

and RAS-associated markers.

Methods and materials

Reagents and cell lines

Anti-ATF2 (ab47476) and anti-pATF2 (ab13106) were

purchased from Abcam (USA). Anti-B cell lymphoma

(BCL)-xL (sc-7195), anti-BCL2-associated X protein

(BAX; sc-493), anti-caspase 3 (sc-7148), and anti-b-actin

(sc-130656) were obtained from Santa Cruz Biotechnology

(CA, USA). Secondary goat anti-rabbit IgG conjugated

with horseradish peroxidase (BA1054) was purchased from

BOSTER (Wuhan, China). The western blot analysis kit

was from Beyotime (China). Real-time PCR reagents and

primers came from TaKaRa (Dalian, China). CFZ (S2853)

and z-VAD-fmk (S7023) were purchased from Selleck

(USA) and resolved with dimethyl sulfoxide. Commer-

cially available murine colon 26 adenocarcinoma cell line

C26 cells were a gift from the Department of Pathology,

Chongqing University of Medical Sciences.

Animal studies

All animal experimental procedures were reviewed and

approved by the Institutional Animal Care and Use Com-

mittee of Chongqing University of Medical Sciences.

Male BALB/C mice (6–8 weeks old; 20–24 g) were

obtained from the Experimental Animal Center of

Chongqing University of Medical Sciences, China. The

animals were housed in a controlled environment with

specific temperature (22 ± 1 �C) and humidity

(55 ± 5 %) settings and a 12-h light–dark cycle; all ani-

mals were fed ad libitum. To acclimate to local conditions,

the animals were housed in the facility for at least 7 days

before the experiments began.

To induce cancer cachexia, C26 cells growing in ex-

ponential phase were harvested with trypsin and injected

subcutaneously into an axilla of the mouse. A total of 175

animals received C26 cell injections with 1 9 106 cells per

site; 10 animals received PBS injection instead of C26 cells

to serve as healthy controls.

The tumor-bearing animals were divided into 7 groups

of 25 animals each, according to the treatment and the time

when the treatment started: CFZ (2 mg/kg, twice a week)

and z-VAD-fmk (1.5 mg/kg, daily), alone (designated as

‘‘C’’ or ‘‘Z,’’ respectively) or in combination (designated as

‘‘U’’). Each of these treatments was administered either

5 days after cell inoculation (preventive), when the tumor

nodules were palpable, or 12 days after cell inoculation

(post-cachexia), when the mice presented signs of

cachexia. In addition, a group of tumor-bearing mice re-

ceived sterile phosphate-buffered saline (PBS) to serve as

the cachexia control (CC); another group of mice received

subcutaneous injection of PBS, instead of C26, were the

healthy controls (HC).

On day 19, 10 animals from each group were killed for

bioanalytical studies; the remaining mice (15 mice from

each group) were used for longitudinal studies of the

spontaneous physical activity and survival.

We recorded spontaneous physical activity, condition of

the fur, and body weight of the animals on a daily basis.

The spontaneous physical activity was monitored by an

infrared monitoring system (WV-CF314LCH, Panasonic,

Japan). In addition, tumor growth was measured by a

caliper, and the tumor volume was determined using the

following formula: tumor volume V (cm3) = (a 9 b2)/2,

where a represents the length in millimeter and b the width

in millimeter.

100 Page 2 of 10 Med Oncol (2015) 32:100

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On day 19, retro-orbital blood samples were collected

from 10 mice in each group. Serum was obtained by

clotting of the blood at room temperature for 1 h followed

by centrifugation at 4000 rpm/min for 10 min. The serum

samples were stored at -20 �C before analyses. After

blood collection, the animals were euthanized by cervical

dislocation; tumors and gastrocnemius muscles from the

left leg were dissected and weighed. Muscle specimens

were divided into two portions, with one snap-frozen at

-80 �C for biochemical analyses and the other fixed in

10 % buffered formalin for histochemical studies.

Histology examination

Five-micron serial sections of paraffin-preserved gastroc-

nemius muscles were stained with hematoxylin and eosin.

The images were acquired under an inverted light micro-

scope (2009) and analyzed with Image J (http://rsb.info.

nih.gov/ij/). Approximately 200 muscle fibers from each

muscle sample were analyzed for cross-sectional area

quantification.

Bioanalytical assays

Serum glucose, triglyceride, total protein, and albumin

were measured using a Beckman counter (USA). Rennin

(sc-22752) and AngII (sc-9040) (santacruze, USA) levels

in serum were determined using sandwich enzyme-linked

immunosorbent assay.

Real-time reverse transcription PCR (qRT-PCR)

analysis

Total RNA was isolated from frozen gastrocnemius muscle

samples with TRIzol reagent (TaKaRa, China) and reverse

transcribed to cDNA in accordance with the manufactur-

er’s instructions. RT-PCR was performed using the SYBR

Green quantitative reverse transcription PCR System

(KAPA Biosystems, USA). Each qRT-PCR reaction

(10 lL) included 1 lL of diluted cDNA, 0.6 lL of primers,

5 lL of KAPA SYBR fast qPCR master mix, and double-

distilled water.

Table 1 contains a list of all the primers used in the real-

time PCR analysis. Real-time-PCR was performed using an

ABI Prism 7500 sequence detection system (Applied

Biosystems), under the following reaction conditions:

95 �C for 1 min, 40 cycles of a three-step reaction, de-

naturation at 95 �C for 5 s, annealing at 58 �C for 15 s, and

extension at 72 �C for 15 s.

Melting curve analyses were performed with the Bio-

Rad CFX Manager v1.6 (Bio-Rad) to ensure amplification

specificity. The amount of the target transcripts were nor-

malized to glyceraldehyde 3-phosphate dehydrogenase

(GAPDH), and the relative fold changes in expression were

calculated using the comparative cycle threshold (Ct)

method.

Western blot analysis

Nuclear and total proteins were extracted from gastrocne-

mius muscles with RIPA buffer (50 mM Tris pH 7.4,

150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxy-

cholate, 0.1 % sodium dodecyl sulfate), and multiple pro-

tease inhibitors (including sodium orthovanadate, sodium

fluoride, ethylenediaminetetraacetic acid, leupeptin), and

quantified with a bicinchoninic acid assay. Protein lysates

were subsequently resolved with 10 % sodium dodecyl

sulfate–PAGE and transferred onto a polyvinylidene di-

fluoride membrane (Beyotime Institute of Biotechnology,

China). The membranes was blocked with 5 % skim milk

at 37 �C for 1 h and then incubated with specific primary

antibodies overnight at 4 �C. Thereafter, the membranes

were washed with Tris-buffered saline with Tween 20 and

incubated with secondary goat anti-rabbit IgG-horseradish

peroxidase for 1 h at room temperature. Proteins of interest

were visualized using a UVP BioImaging system (Super

bright scientific instrument co., LTD, New York, America)

and analyzed with LabWorks software (Gene co., LTD.,

Shanghai, China).

Statistical analyses

SPSS 17.0 was used for statistical analyses, and all data are

presented as mean ± standard deviation. One-way

ANOVA was used to compare more than two groups;

Newman–Keuls analyses were employed for pairwise

Table 1 Primers for real-time PCR

Gene Primer sequence

ATF2 50-GCCCTTCCTCTCCTCAACCA-30

50-AGTCCTAACCAATCCACTGCCA-30

Caspase3 50-GGACTGATGAGGAGATGGCTTG-30

50-AGGGACTGGATGAACCACGAC-30

BAX 50-GGATGCGTCCACCAAGAAGC-30

50-AAAGTAGAAGAGGGCAACCACG-30

BCL-XL 50-GGTAGTGAATGAACTCTTTCGGGA-30

50-CATCTCCTTGTCTACGCTTTCCAC-30

MAFbx 50-GAAGAGAGCAGTATGGGGTCAC-30

50-CTTGAGGGGAAAGRGAGACG-30

MuRFl 50-GGAACACGAAGACGAGAAAATC-30

50-TGGCTATTCTCCTTGGTCACTC-30

GAPDH 50-GGTGAAGGTCGGTGTGAACG-30

50-CTCGCTCCTGGAAGATGGTG-30

Med Oncol (2015) 32:100 Page 3 of 10 100

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comparisons. Survival data were analyzed using a log-rank

test. P \ 0.05 was considered statistically significant.

Results

Combined treatments of CFZ and z-VAD-fmk

ameliorate weight loss and reduce tumor burden

in the C26 tumor-bearing mice

Mice bearing subcutaneous C26 tumors are commonly

used as a rodent model of cancer-associated cachexia [20],

since these mice undergo lethal wasting as the tumors

grow. To evaluate the effect of CFZ and z-VAD-fmk on

cancer-associated cachexia, we administered the two drugs

to the C26-inoculated mice at 5 and 12 days after

inoculation and evaluated changes in body weight, gas-

trocnemius muscle mass, and tumor growth at 5-, 12-, and

19-day post-inoculation.

The body mass of animals in each of the cachexia model

groups was not significantly changed 5 days after C26 cell

inoculation (P [ 0.05; Fig. 1). However, on day 12, the

mean weights of those in the groups treated 5 days after

inoculation with CFZ (CP), z-VAD-fmk (ZP), or CFZ ?

z-VAD-fmk (UP) were significantly higher than those of

the mice treated with PBS only (CC). On day 19, mice in

all groups that had been treated with CFZ, z-VAD-fmk, or

CFZ ? z-VAD-fmk, whether at 5- or 12-day post-

inoculation, had body weights higher than those in the CC

group, but lower than those of the healthy control (HC)

group who did not receive C26 cells.

In addition to body weight, the weight and cross-sec-

tional area of the gastrocnemius muscles of the treatment

groups were also higher than those in the CC group, but

lower than those in the HC group (Table 2). The tumor

burden (weight and volume) of the treated groups was

lower than that of the CC group, with the groups treated at

5-day post-inoculation having a lower tumor burden than

those treated at 12 days. Furthermore, CFZ was more ef-

fective in inhibiting tumor growth than z-VAD-fmk

(Table 2). Overall, the drug administrations at day 5 were

more effective than those performed on day 12, and

CFZ ? z-VAD-fmk treatments were more effective than

either drug given alone (Fig. 1; Table 2).

In the aggregate, these results show that the treatments

with CFZ and z-VAD-fmk, especially the combined earlier

treatment, can ameliorate cancer-associated cachexia.

Combined treatments of CFZ and z-VAD-fmk increase

the spontaneous physical activity and improve survival

of the C26 tumor-bearing mice

To evaluate the effects of CFZ and z-VAD-fmk treatments

on the quality of life and survival of C26 tumor-bearing

mice, we measured the spontaneous physical activity and

lifespan of the animals in the different treatment groups.

Compared with the CC group, the groups treated with CFZ,

z-VAD-fmk, or CFZ ? z-VAD-fmk 5 days after inocula-

tion had significantly greater spontaneous activity

(Fig. 2a). For both model groups treated at 5 or 12 days,

the associated effects of the mono-treatments were less

than those of the combined treatment, and the associated

effect of CFZ was consistently stronger than that of

z-VAD-fmk.

By constructing survival curves with 15 animals in each

treatment or control group, we found that the survival of

groups treated at 5-day post-inoculation was longer than

those groups treated on day 12, but in both cases, survival

was longer in the CFZ-treated mice than the z-VAD-fmk-

treated, and longer in the CFZ ? z-VAD-fmk groups than

in those given either drug alone.

Together, these results show that the treatments with

CFZ and z-VAD-fmk, especially the early combined

treatment, can increase spontaneous physical activity and

prolong the lifespan in mice with cancer-associated

cachexia.

Combined treatments of CFZ and z-VAD-fmk restore

metabolism of the C26 tumor-bearing mice

To evaluate the effect of CFZ and z-VAD-fmk treatments

on the metabolic state of mice with cachexia, we measured

the serum levels of glucose, triglyceride fatty acids, albu-

min, and total proteins in the control and tumor-bearing

Fig. 1 Body weight changes of cachexia model and HC mice. No

difference was observed among the various groups on day 0 and 5

after cell inoculation. On day 12, no significant difference was

detected among groups treated 5 days after inoculation (CP, ZP and

UP), nor among those treated 12 days after inoculation (CT, ZT, UT

and CC). On day 19, the CP and ZP groups were similar, and the CT

and ZT groups were also comparable

100 Page 4 of 10 Med Oncol (2015) 32:100

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groups 19 days after C26 cell inoculation. Compared with

the HC group, all the tumor-bearing mice had lower levels

of glucose, albumin, and total proteins, and higher levels of

triglyceride fatty acids (Table 3). The CFZ and z-VAD-

fmk treatments administered either 5- or 12-day post-

inoculation were associated with higher levels of glucose,

albumin, and total proteins and lower levels of triglyceride

fatty acid, compared with the group given PBS. Consistent

with our above observation in the physiological examina-

tion, survival analysis, and tumor measurement, the pre-

ventive setting was more effective than the post-cachexia

setting; the effect of CFZ treatment was consistently

stronger than that of z-VAD-fmk; the combined treatment

was more effective than the mono-treatments in both set-

tings (Fig. 2a). Together, these results demonstrated that

the treatments with CFA and z-VAD-fmk, especially the

combined treatment in the prevention setting, can reverse

the catabolic state in animals with cachexia.

Treatments of CFZ and z-VAD-fmk alter RAS in C26

tumor-bearing mice

Previous studies [14] have shown that activation of RAS is

associated with cachexia, and ANGII can activate proteins

involved in proteolysis. To test whether treatments with

CFZ and z-VAD-fmk affect RAS activity in mice with

cachexia, we measured the levels of renin and ANGII in

the different experimental groups. Compared with the HC

group, the tumor-bearing animals had much higher levels

of renin and ANGII. Treatments with CFZ and z-VAD-fmk

were associated with lower levels of renin and ANGII. The

renin and ANGII levels of the CFZ-treated groups were

lower than those of the z-VAD-fmk-treated group, and

lower still in the CFZ ? z-VAD-fmk group than those

receiving either drug alone. The renin and ANGII levels of

the group treated with CFZ 5 days after inoculation were

lower than those of the CFZ group treated at 12 days, but

no difference was observed between the groups receiving

z-VAD-fmk treatments at the two times (Fig. 3a, b).

To explore the molecular mechanism underlying CFZ and

z-VAD-fmk-associated alteration in RAS, we measured the

levels of ATF2 in the gastrocnemius muscles of animals in

the different groups. Results from both qRT-PCR and

western blot analyses showed that ATF2 levels were higher

in the tumor-bearing animals than in the healthy controls

(Fig. 3c, d; Table 4). ATF2 levels in the groups treated with

CFZ, z-VAD-fmk, or CFZ ? z-VAD-fmk, at either 5 or

12 days after inoculation, were lower than in the CC group

(treated with PBS). There was no difference in ATF2 levels

between corresponding groups treated at 5 and 12 days.

Regarding the levels of pATF2, the active form of ATF2,

these were dramatically higher in the CFZ-treated groups

(CFZ-only and CFZ ? z-VAD-fmk) than in the z-VAD-fmkTa

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Med Oncol (2015) 32:100 Page 5 of 10 100

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Page 6

groups, with correspondingly higher ratios of pATF2 to

ATF2 (Fig. 3d, e). Since high ATF2 activity can inhibit renin

transcription, this result suggested a possible mechanism by

which CFZ inhibits RAS activity.

Treatments of CFZ and z-VAD-fmk inhibit apoptosis

and the ubiquitin-proteolysis pathways

To characterize the effect of CFZ and z-VAD-fmk on

apoptosis and the UPS, we measured the expression of

several well-characterized genes involved in the pathways,

including caspase 3, BAX, and BCL-XL for apoptosis, and

MuRF1 and MAFbx for the ubiquitin degradation pathway.

Meanwhile, we measured the levels of caspase 3, BAX,

and BCL-XL proteins. Results from both qRT-PCR and

western blot analyses revealed that treatments with CFZ

and z-VAD-fmk were associated with an inhibition of the

expression of caspase 3 and BAX and induced the ex-

pression of BCL-XL, resulting in a decrease in the BAX-

to-BCL-XL ratio (Fig. 4a–e). The CFZ ? z-VAD-fmk

treatments (at 5 and 12 days) had a greater effect than the

corresponding mono-treatments; treatments administered

5 days after inoculation were more effective than those

administered at 12 days; no differences were observed

between the CFZ and z-VAD-fmk groups. Similarly, both

CFZ and z-VAD-fmk treatments inhibited the expression

of MuRF1 and MAFbxm, two genes involved in ubiquitin-

mediated protein degradation pathway. The inhibitory ef-

fect of CFZ was stronger than that of z-VAD-fmk (Fig. 4e,

f). These results suggested that CFZ and z-VAD-fmk may

ameliorate cachexia via inhibiting apoptosis and ubiquitin-

medicated protein degradation pathway.

Fig. 2 The spontaneous physical activity and overall survival of the

C26 tumor-bearing mice treated with CFZ, z-VAD-fmk, or CFZ ? z-

VAD-fmk at 5- or 12-day post-inoculation and control mice. a The

spontaneous physical activities of animals in different treatment

groups. The P values between most of the group pairs were \0.001,

except for P \ 0.05 between CP and UT (*) and between ZP and CT

(D). b Kaplan–Meier survival curves of animals in different treatment

groups. The P values between most of the group pairs were \0.001,

except for P \ 0.05 between CP and UT (#) and between ZP and CT

(&)

Table 3 Metabolic parameters of the experimental groups at 19 days

Treated 5-day post-inoculation Treated 12-day post-inoculation

HC CC CP ZP UP CT ZT UT

Serum

glucose,

mmol/L

5.99 ± 0.26 3.06 ± 0.18 4.71 ± 0.171 3.95 ± 0.182 4.94 ± 0.55 4.17 ± 0.172 3.48 ± 0.14 4.53 ± 0.171

Serum

TFA,

mmol/L

1.16 ± 0.15 4.99 ± 0.32 2.93 ± 0.181 3.60 ± 0.222 2.26 ± 0.20 3.75 ± 0.282 4.29 ± 0.31 3.13 ± 0.171

Albumin,

g/L

19.84 ± 0.47 13.23 ± 0.28 16.45 ± 0.291 14.96 ± 0.322 18.43 ± 0.35 15.26 ± 0.412 14.15 ± 0.19 16.29 ± 0.431

Total

protein,

g/L

59.17 ± 0.90 49.21 ± 1.21 56.61 ± 1.081 54.07 ± 1.222 57.83 ± 1.18 55.13 ± 0.942,3 52.28 ± 1.29 55.91 ± 1.381,3

The difference between most of the groups was statistically significant (P \ 0.05), with the exception of the groups with the same superscripted

numbers. TFA triglyceride fatty acid

100 Page 6 of 10 Med Oncol (2015) 32:100

123

Page 7

Discussion

Cancer cachexia is a common and detrimental syndrome in

patients with cancer. It is associated with increased treat-

ment toxicity and decreased quality of life in cancer pa-

tients and accounts for over 30 % of cancer-related deaths

[21]. Despite its significant impact on the morbidity and

mortality in patients, no effective therapies have been de-

veloped. Given that skeletal muscle wasting is the key

symptom of cancer cachexia, and caspase 3 and UPS are

essential to skeletal muscle proteolysis, we reasoned that

concurrent inhibition of caspase 3 and UPS may be an

effective treatment for cancer cachexia.

In the current study, we tested the efficacy of CFZ, a

novel irreversible 20S proteasome inhibitor, and z-VAD-

fmk, a broad-spectrum caspase inhibitor, in alleviating

cancer cachexia in the well-established C26 mouse model

for cancer cachexia. We demonstrated that treatments with

CFZ and z-VAD-fmk prevented muscle wasting, reduced

tumor burden, modulated metabolism, and increased the

spontaneous physical activity of the mice with cachexia.

Furthermore, our results showed that combined treatment

in the preventive setting exhibited the highest efficacy.

These results suggest that combined treatment with CFZ

and z-VAD-fmk may be a promising option for patients

with cancer-associated cachexia and warrants further

investigation.

The role of apoptosis in cachexia has been well estab-

lished. Elevated levels of apoptotic proteins were detected

in the skeletal muscle of tumor-bearing rabbits [22], and

apoptosis of non-diseased organs in these rabbits led to

multiple organ failure [23]. While BCL-2 and BCL-XL

have anti-apoptotic activity, BAX is a pro-apoptotic protein

[24]. In the present study, we observed higher levels of

Fig. 3 Effect on the RAS of

CFZ, z-VAD-fmk, or CFZ ?

z-VAD-fmk treatments

administered 5 or 12 days after

C26 inoculation and control

groups. Serum levels of a renin

and b ANGII. c The mRNA

expression of ATF2. d Western

blot analysis of ATF2 and

pATF2. e pATF2-to-ATF2

ratio. Significant differences

were detected between most of

the groups, with the exceptions

of (A and B) CP and UT (D) and

ZP and ZT (#); and (C and E)

CP and UP (*), CT and UT (#),

and HC, ZP, ZT and CC (&)

Med Oncol (2015) 32:100 Page 7 of 10 100

123

Page 8

BAX and caspase 3 and lower levels of BCL-XL in mice

with cachexia, and we also found that CFZ and z-VAD-

fmk treatments were associated with lower levels of cas-

pase 3 and BAX and higher levels of BCL-XL, leading to a

lower BAX-to-BCL-XL ratio than in PBS-treated cachexia

mice. These observations suggest that these treatments

inhibited skeletal muscle apoptosis. Since caspase 3 also

serves as the initial activator in proteolysis, the lower

levels of caspase 3 also provide an explanation for the

lesser amount of muscle proteolysis observed in cachexia

mice treated with CFZ and z-VAD-fmk.

RAS has an important role in cachexia, and ANGII

levels were found to be higher in patients with cachexia

[9, 10, 25]. ANGII can promote the E3 ligases MuRFl and

MAFbx, inducing proteolysis of skeletal muscle [26].

ANGII can also activate caspase 3 [25]. Increased levels

of MuRF-1 and MAFbx via ANGII have been suggested

as the underlying mechanism for ANG II-induced skeletal

muscle atrophy in rodents [12]. In the present study, we

found that mice treated with CFZ and z-VAD-fmk had

lower levels of ANGII, MuRF1, and MAFbx. Therefore, it

is possible that the effect of CFZ and z-VAD-fmk is

mediated in part through the downregulation of ANGII

levels, which subsequently lowers levels of MuRF-1 and

MAFbx.

In the RAS, renin is the rate-limiting step for the con-

version of angiotensinogen to ANGI. Therefore, inhibiting

renin expression can restrict levels of ANGI and subse-

quently that of ANGII. In the current study, we found that

the combined treatment of CFZ and z-VAD-fmk led to

lower renin levels. This provides an explanation for the

lower ANGII levels found in these mice. The cAMP re-

sponse element (enhCRE) in the distal enhancer regulatory

region of the renin gene has been reported to be important

in controlling renin transcription. enhCRE is also a binding

target of ATF2: Once activated by phosphorylation, ATF2

binds to enhCRE to replace the CRE-binding protein

(CREB), causing suppression in renin expression [13, 27].

In the present study, we observed higher pATF2 levels in

the CFZ-treated animals but not the other groups. The high

pATF2 in CFZ-treated animals could be the result of the

inhibition of UPS by CFZ. The higher pATF2 levels lead

to a higher pATF2-to-ATF2 ratio, providing a plausible

explanation for the decrease in renin expression in CFZ-

treated mice with cachexia.

It is worth noting that z-VAD-fmk treatment was not

associated with higher pATF2 levels. z-VAD-fmk is an

apoptosis inhibitor and is not expected to increase pATF2

levels by downregulating proteasome activity. The fact that

z-VAD-fmk treatment caused no pATF2 upregulation

suggests that downregulation of RAS by pATF2 is specific

to CFZ treatment, and other mechanisms are involved in

z-VAD-fmk-mediated cachexia rescue.Ta

ble

4P

rote

inle

vel

so

fA

TF

2,

pA

TF

2,

casp

ase

3,

BA

X,

and

BC

L-X

Lin

the

gas

tro

cnem

ius

mu

scle

so

fm

ice

Tre

ated

5-d

ayp

ost

-in

ocu

lati

on

Tre

ated

12

-day

po

st-i

no

cula

tio

n

HC

CC

CP

ZP

UP

CT

ZT

UT

AT

F2

0.4

3±

0.0

90

.97

±0

.11

0.6

3±

0.0

81

0.6

9±

0.0

81

0.6

5±

0.0

51

0.8

5±

0.0

52

0.8

4±

0.0

52

0.8

2±

0.0

52

pA

TF

20

.19

±0

.06

30

.39

±0

.06

60

.58

±0

.08

40

.27

±0

.06

3,5

0.6

3±

0.0

54

0.5

4±

0.0

84

0.3

2±

0.0

35,6

0.5

4±

0.0

74

Cas

pas

e3

0.3

7±

0.0

40

.97

±0

.11

0.6

6±

0.0

77

0.6

3±

0.0

57

0.5

1±

0.0

90

.83

±0

.04

80

.78

±0

.05

80

.65

±0

.09

7

BA

X0

.23

±0

.03

0.7

0±

0.0

40

.43

±0

.02

90

.41

±0

.02

90

.32

±0

.06

0.6

0±

0.0

910

0.5

7±

0.0

610

0.4

6±

0.0

69

BC

L-X

L0

.60

±0

.07

11

0.3

8±

0.0

50

.73

±0

.03

12

0.6

7±

0.0

411,1

20

.84

±0

.04

0.5

5±

0.0

611,1

30

.50

±0

.03

13

0.6

7±

0.0

711,1

2

No

rmal

ized

tob

-act

in.

Th

ed

iffe

ren

ces

bet

wee

nm

ost

of

the

gro

up

sw

ere

sig

nifi

can

t(P

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),w

ith

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s

100 Page 8 of 10 Med Oncol (2015) 32:100

123

Page 9

Fig. 4 Treatments with CFZ,

z-VAD-fmk, and CFZ ?

z-VAD-fmk altered the

expression of genes involved in

apoptosis and the UPS in

gastrocnemius muscles. (A to C)

The mRNA expression levels of

a caspase 3, b BCL-XL, and

c Bax. d The ratio of Bax to

BCL-XL. e Caspase 3, BCL-

XL, and BAX proteins. The

mRNA expressions of

(f) MuRF1 and (g) MAFbx.

Significant differences were

detected between most of the

groups with the exceptions of

those whose labels are the same

in the graph. For example, there

is no statistically significant

difference between HC, ZP, and

UT (labeled with symbol ‘‘$’’)

and no difference between CT

and ZT (labeled with ‘‘¥’’) in

panel A

Med Oncol (2015) 32:100 Page 9 of 10 100

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Page 10

In the aggregate, we found that treatments with CFZ and

z-VAD-fmk were effective in preventing weight loss,

muscle atrophy, and tumor growth and increased the

quality of life and survival in mice with cancer-associated

cachexia. In particular, we found that the early combined

treatment of these drugs was more effective than the other

treatment options. Our molecular exploration suggests that

CFZ and z-VAD-fmk treatments can inhibit apoptosis and

the proteolysis pathways in skeletal muscles. Furthermore,

the RAS system may be involved in downregulating pro-

teolytic proteins in the UPS, and regulation of ATF2 ac-

tivity may be involved in regulating RAS in cachexia

model mice treated with CFZ. These results warrant further

evaluation of the viability of administering combined CFZ

and z-VAD-fmk early in the treatment of cancer patients

with cachexia.

Acknowledgments This work was supported by the Scientific Re-

search Project of Chongqing Health Bureau, China (Grant number:

2011-2-101). Hua Tang and Qiang Wang designed and executed the

study. Chunhong Li cultured the cells used in the study. Qiang Wang

is responsible for all the statistical analysis and manuscript writing.

All the authors participated in the development of the animal model

and the revision of the manuscript.

Conflict of interest The authors declare no conflict of interest.

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