`Advanced
`
`Pharmaceutical
`
`
`
`Bulletin
`
`Adv Pharm Bull, 2014, 4(Suppl 1), 465-470
`doi: 10.5681/apb.2014.069
`http://apb.tbzmed.ac.ir
`
`Research Article
`in
`Immunomodulatory Compounds
`Effects of Some Natural
`Combination with Thalidomide on Survival Rate and Tumor Size in
`Fibrosarcoma-Bearing Mice
`
`Reza Aghebati Maleki1, Dariush Shanehbandi2, Saeed Sadigh Eteghad3, Habib Zarredar2, Fatemeh Zare
`Shahneh2, Leili Aghebati Maleki2, Mehrnosh Samavati2, Hamed Asadi1, Seyed Ehsan Mosavi1, Afshin
`Habibzadeh1, Mozhdeh Mohammadian4, Behzad Baradaran2*
`
`1 Hematology and Oncology Research Center, Tabriz University of Medical sciences, Tabriz, Iran.
`2 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
`3 Neurosciences Research Center (NSRC), Tabriz University of Medical Sciences, Tabriz, Iran.
`4Amol Faculty of Paramedical Sciences, Mazandaran University of Medical Sciences, Sari, Iran.
`
`
`
`
`
`
`
`Article History:
`Received: 31 May 2014
`Revised: 14 June 2014
`Accepted: 18 June 2014
`ePublished: 25 August 2014
`
`Keywords:
`
` Fibrosarcoma
` Thalidomide
`
` HSP
` BCG
`
` Bifidobacterium
` IMOD
`
`
`
`
`
`
`
`Abstract
`Purpose: Despite significant advances have been achieved in cancer therapy, response to
`conventional treatments like surgery, radiotherapy and chemotherapy varies among
`individuals. Immunotherapy is known to be an effective strategy for patients who are
`resistant to the currently available interventions.
`Methods: Ninety-six male Balb/c mice (aged 6-8 weeks) were selected and divided into
`twelve groups of eight. Approximately, 1×106of WEHI-164 cells were injected to each
`mouse for tumor genesis. Five immunotherapy treatments were considered in this study,
`including Heat Shock Proteins (HSP), Bacillus Calmette-Guérin (BCG), Bifidobacterium,
`Immuno-Modulator Drug (IMOD) and Thalidomide. After tumor formation, the groups
`were treated with one or more of these therapies. Tumor size and survival rate was regularly
`monitored.
`Results: Depending on the treatment group, tumor sizes were different. In some groups,
`combined treatments demonstrated more inhibitory effects on tumor growth rate. The mice
`in group (IMOD+ Thalidomide) had the lowest survival rate but group (BCG+ HSP+
`Thalidomide) survived until the end of the experiment.
`Conclusion: The (HSP+ BCG+ Thalidomide) group exhibited satisfactory outcomes and
`two third of the mice in this group went into complete remission. Some combination
`therapies in test groups had significant impacts on survival and tumor growth rate.
`
`
`Introduction
`that utilize
`strategies
`Nowadays, multidisciplinary
`surgery, chemotherapy and radiation have drastically
`improved the survival rate of the patients suffering from
`cancer.1 However, since many patients are resistant to
`conventional
`therapies
`the mentioned malignancy
`remains a leading cause of mortality worldwide.2 On the
`contrary, immunotherapy represents a variety of effective
`applications to improve overall survival and decrease
`treatment- associated morbidity.3,4 In this regard, we
`have assessed the efficacy of some immunotherapy
`approaches, alone and combination with each other, in
`mouse tumor models.
`HSPs are intracellular molecules that act as chaperones
`for antigens.5 From the immunological point of view,
`HSPs are involved in stimulation of the innate and
`adaptive immune systems. The ability of HSPs in
`binding to the antigenic peptides and transporting them
`to the antigen presenting cells (APC) on MHC-I is the
`basis for their potential role in the generation of peptide-
`
`specific T lymphocyte responses.6,7 HSPs, as anti-tumor
`vaccines are involved in phase II and III clinical trials for
`cancer immunotherapy.8,9
`The chemical composition of Bacillus Calmette-cell
`Guérin walls (BCG-CW) demonstrates immunoadjuvant
`and anti-tumor activities.10 The activation of immune
`cells and dendritic cells (DC) and the identification of the
`receptors on the surface of DC for BCG-CW are
`responsible for its immunological activities.11 BCG-
`CWS up-regulates a maturation marker, CD83, and co-
`stimulators, CD80 and CD86, as well as MHC levels,
`and secretion of IL-12 and TNF-α by DC.12
`In general,
`tumor progression
`is accompanied by
`angiogenic factors like VEGF, bFGF and TGF-β.13 These
`factors could be arrested by Thalidomide, Thiols, and
`components.14,15
`Polyphenolic
`In
`other words,
`Thalidomide plays its role by prohibiting the formation
`of new vascular network from existing blood vessels.16,17
`By this mechanism, Thalidomide can delay oxygen and
`
`*Corresponding author: Behzad Baradaran, Tel: +98 (411) 3364665, Emails: baradaranb@tbzmed.ac.ir, behzad_im@yahoo.com
`©2014 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits
`unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from
`the authors or the publishers.
`
`Genome Ex. 1048
`Page 1 of 6
`
`

`

`
`
`Aghebati Maleki et al.
`
`to
`
`tumors and restrain
`
`the cell
`
`nutrients delivery
`division.18
`Bifidobacteria are gram-positive, obligate an aerobes
`naturally found in large intestine and the lower small
`intestine of human and some mammals.19 These non-
`pathogenic bacteria show inhibitory effects on colon,
`mammary, liver and HPV-associated cervicalcancers.20,21
`Bifidobacteria
`can
`only
`proliferate
`in
`anoxic
`environments and thus preferentially tend to accumulate
`in such regions of solid tumors.17 For this reason, they
`could also be employed in cancer gene therapy.22
`Immuno-Modulator Drug (IMOD) is an anti-AIDS drug,
`which is composed of two chemical components and
`three herbal extracts.23 It is shown that, this drug has
`immuno- modulatory and anti-AIDS effects.23,24
`In this study, we have tried to find an appropriate
`combination of the aforementioned strategies to prevent
`the invasive cancer, to decrease the tumor size and
`increase the survival rates.
`
`Materials and Methods
`Study design
`Ninety-six male inbred Balb/c mice, aged from six to
`eight weeks, were purchased from Pasture Institute
`(Tehran, Iran) and housed under conditions of constant
`temperature and humidity according to institutional
`ethical guidelines. The mice were randomly divided into
`12 groups of eight as following: 1: Control, 2: BCG, 3:
`HSP, 4: Thalidomide, 5: Bifidobacterium, 6: IMOD, 7:
`BCG+ Thalidomide, 8: IMOD+ Bifidobacterium, 9:
`IMOD+ HSP, 10: IMOD+ Thalidomide, 11: HSP+
`BCG+ Thalidomide and 12: IMOD+ Bifidobacterium+
`HSP+ BCG+ Thalidomide. Approximately 1×106
`cells/100µl of WEHI-164 cells were injected to create
`fibrosarcoma tumors in mice. Consequently, tumors were
`generated after 11 days.
`
`Treatment method
`the
`information about
`Table 1 represents detailed
`medication groups. Drugs were Injected twice a week
`from the 11th day intratumorally and subcutaneously. In
`control group, 200 μl of PBS was injected to mice. The
`BCG used for injection was diluted with PBS in a ratio of
`1:1. In HSP group, WEHI-164 cells were first stimulated
`to produce HSP. To release HSP, WEHI-164 cells were
`cultivated in flasks and incubated in a 42°C water bath for
`60 min. The cells were
`trypsinated after 12-hour
`incubation at 37°C and washed thrice with PBS. Finally,
`cell suspension containing 5×105 cells/100µlin PBS was
`prepared. Cell lysates were produced by five times freeze
`and thaw and HSP-70 expression was detected using
`western blotting assay. For this purpose, equal amounts of
`lysate proteins (extracted from 5×105 WEHI-164 cells)
`were fractionated by 10% SDS-PAGE gels and transferred
`into a polyvinylidene fluoride (PVDF) membrane. After
`being washed, the membrane was blocked with 5% skim
`milk at room temperature for 1h and incubated with the
`mouse monoclonal antibody against HSP-70 (1:1000
`R&D systems) at 4°C overnight. After washing, the
`membrane was incubated with horseradish peroxidase-
`conjugated anti-mouse antibody (1:1000, Sigma) at room
`temperature for 1h. Finally, the immune-reactive bands
`were detected by ECL (Amersham Phamacia Biotech Inc,
`USA) (Figure 1). After
`the confirmation of HSP
`production,
`lysates were
`injected
`twice a week,
`intratumorally and subcutaneously. Thalidomide was
`solved
`in DMSO/PBS
`solvent according
`to
`the
`manufacturer’s
`recommendations. The
`lyophilized
`Bifidobacteria were solved in sterile PBS for injection.
`The bacterial suspension was prepared in 1:10 ratio.
`IMOD was purchased from Pars Roos Company, Tehran-
`Iran and diluted in a ratio of 1:10 with 5% dextrose.23
`
`Table 1. Study groups and prescribed treatment plans
`
`
`
`
`
`
`
`Treatments
`
`
`
`BCG
`
`
`
`HSP
`
`
`
`Thalid.
`
`
`
`Bifidobacter.
`
`
`
`IMOD
`
`1- Control
`2- BCG
`3- HSP
`4- Thalidomide
`5- Bifidobacterium
`6- IMOD
`7- BCG+ Thalidomide
`8- IMOD+ Bifidobacterium
`9- IMOD+ HSP
`10- IMOD+ Thalidomide
`11- HSP+ BCG+ Thalidomide
`12- IMOD + Bifidobacterium + HSP +
`BCG + Thalidomide
`
`-
`-
`200 µl
`-
`-
`-
`-
`-
`100 µl
`-
`100 µl
`
`100 µl
`
`
`
`
`
`
`
`
`
`-
`200 µl
`-
`-
`-
`-
`200 µl
`-
`-
`-
`100 µl
`
`100 µl
`
`
`
`
`
`
`
`
`
`
`
`-
`-
`-
`200 µl
`-
`-
`100 µl
`-
`-
`100 µl
`100 µl
`
`100 µl
`
`
`
`
`
`
`
`
`
`
`-
`-
`-
`-
`200 µl
`-
`-
`100 µl
`-
`-
`-
`
`
`
`
`
`-
`-
`-
`-
`-
`200 µl
`-
`200 µl
`150 µl
`200 µl
`-
`
`
`
`
`
`
`
`
`
`100 µl
`
`
`
`100 µl
`
`Tumors size
`The size of tumors was measured weekly on special day
`and the volume was calculated by the following formula:
`
`Tumor volume in mm3 = (width) 2 × (length) × π/6. The
`survey of tumors was continued until the end of the 14th
`week.
`
` 466 | Advanced Pharmaceutical Bulletin, 2014, 4(Suppl 1), 465-470
`
`Genome Ex. 1048
`Page 2 of 6
`
`

`

`
`
`Effects of Some Immunomodulators on Survival and Tumor Size
`
`
`Figure 1. HSP-70 expression of heat shocked tumor cells.
`Western blot analysis of HSP-70 expression in WEHI-164 cells,
`maintained at 37°C (2), heat treated 42°C, 60 min, (12 h
`recovery in 37 °C) (1).
`
`Statistical analysis
`One-way, two-way, or repeated measures ANOVA were
`used to identify significant group differences. To assess
`survival rates, the Kaplan-Meier model was used. Values
`of p< 0.05 were considered significant in all studies, and
`all p values were two-tailed.
`
`Results
`Despite the fact that, all mice were injected with the
`same dose of WEHI-164 cells, one mouse in each of
`following groups including HSP, Thalidomide, IMOD
`+ Bifidobacterium, IMOD+HSP and Thalidomide+
`IMOD groups did not developed any fibrosarcoma
`tumors and excluded from the study. According to the
`results, tumor size differed among the treatment groups,
`dependent on the drugs administered. Mean sizes of
`tumors are also represented in Table 2. Table 3,
`indicates survival situation among the test groups. The
`Maximum tumor size was observed in HSP group. In
`comparison with
`the control group, a significant
`decrease
`in
`tumor size was evident
`in BCG +
`Thalidomide group and 25% of the mice could survive
`until week 13. In the IMOD + Bifidobacterium group,
`the tumor growth rate was faster than the control group
`and survival rate was lower as well. In Thalidomide+
`
`IMOD group, tumors grew faster than the control
`group. The best inhibitory effect on tumor size was
`seen in HSP + BCG + Thalidomide group and the mice
`survived until the end of the experiment (Figure 2). The
`tumor growth in this group was slower than the control
`group and a reduction in tumor size was noticeable
`from the7th and 8th weeks.
`
`
`
`
`in BCG, HSP and
`Figure 2. Comparison of survival
`Thalidomide
`(combination
`therapy) and control groups.
`Despite control group, most of the mice in BCG, HSP and
`Thalidomide group went into complete remission.
`
`
`
`
`
`Control
`
`BCG
`
`HSP
`
`
`Table 2. Means of tumor sizes in various groups during the experiment.
`
`Weeks
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`191.6±12
`
`457±32
`
`592.3±49
`
`698.3±47
`
`931.3±98
`
`1710±123 2680±187 3990±405
`
`4688±308
`
`34.3±9
`
`184.6±17
`
`637.6±14
`
`1495.3±108 2594.6±180 3943.3±185 5006.6±186 5926±205
`
`6304±189
`
`445.3±34 791.6±50
`
`1326.3±94
`
`1997.3±124 2573.3±145 2973±110 4582.6±354 6568.6±240 7812±217
`
`Thalidomid
`
`280±34
`
`469.3±42
`
`610.6±84
`
`769.6±94 1586.3±104 2829±131 3971±158 4191.5±284 5570±291
`
`10
`
`NA
`
`NA
`
`NA
`
`NA
`
`11
`
`NA
`
`NA
`
`NA
`
`NA
`
`243.3±39
`
`557±45
`
`1069±91
`
`1573±106 2267.3±187 1823±128 3324.3±204 4397.3±315 4480±301 4605±294
`
`NA
`
`12
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`13
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`14
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`Bifidobacterium
`
`IMOD
`
`175.6±14
`
`243±12
`
`537.6±45
`
`909.3±54
`
`1521±94 2398.6±170 3421.6±182 3848±204
`
`4720±208 4156±267
`
`NA
`
`NA
`
`BCG & Thalidomid
`
`221.0±24 671.6±80
`
`1093.6±102 1423.6±134 1208.3±158 1916.3±142 2033.3±245 2073.3±214 2171.3±194 2100±218 2149±187
`
`NA
`
`IMOD & Bifidobacterium
`
`194.6±34 589.3±56
`
`1218±78
`
`1847±84 2974.6±107 5619±204
`
`NA
`
`NA
`
`IMOD & HSP
`
`237.6±45
`
`422±34
`
`794.6±84
`
`1130±108 2784±278 4152.6±207 5053±324 6332±219
`
`IMOD & Thalidomid
`
`535.3±67 979.3±85
`
`1539.6±120 2749.6±240 4445±290
`
`NA
`
`NA±
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`NA
`
`BCG, HSP & Thalidomid
`
`148.3±12 203.3±19
`
`331.6±45
`
`416.3±52
`
`420.1±43
`
`411.3±32
`
`234±34*
`
`286.3±27*
`
`294.3±20* 218.3±22# 206±17# 172.6±19# 156±14# 57±14#
`
`BCG, HSP, Thalidomid,
`IMOD & Bifidobacterium
`
`215.6±27 515.6±35
`
`666.3±74
`
`987.3±86 996.3±102
`
`1397±94 2252.6±145 2703.3±120 3116.6±124 3088±118 2700±126
`
`NA
`
`NA
`
`NA
`
`* Significant reduction in tumor size compared to control group.
`# not compared because of unavailability of control group.
`NA: Not Assessed.
`
`Advanced Pharmaceutical Bulletin, 2014, 4(Suppl 1), 465-470
`
`| 467
`
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`

`
`
`Aghebati Maleki et al.
`
`Table 3. Survival rate in various test groups during the experiment
`
`1 2 3 4 5 6 7 8 9 10 11 12 13 14
`
`Weeks
`
`8 8 8 8 7 7 7 6 3
`
`8 8 7 7 7 7 6 5 3
`
`7 6 6 6 5 5 4 3 2
`
`7 7 7 7 6 6 6 5 3
`
`8 7 7 6 5 5 4 3 3
`
`8 8 8 7 6 5 4 4 3
`
`0
`
`0
`
`0
`
`0
`
`3
`
`3
`
`3
`
`0
`
`0
`
`0
`
`5
`
`3
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`2
`
`0
`
`0
`
`0
`
`5
`
`2
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`5
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`5
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`5
`
`0
`
`
`
`Control
`
`BCG
`
`HSP
`
`Thalidomid
`
`Bifidobacterium
`
`IMOD
`
`BCG & Thalidomid
`
`8 8 7 6 6 5 5 4 4
`
`IMOD & Bifidobacterium
`
`7 7 6 4 3 2 0 0 0
`
`IMOD & HSP
`
`7 7 6 5 4 3 3 2 0
`
`IMOD & Thalidomid
`
`7 5 4 3 2 0 0 0 0
`
`BCG, HSP & Thalidomid
`BCG, HSP, Thalidomid, IMOD &
`Bifidobacterium
`
`8 8 8 7 6 6 5 5 5
`
`8 8 7 7 5 5 4 4 3
`
`
`
`Discussion
`In most treatments used currently, only certain aspects of
`cancer, which overcome
`the
`immune system
`is
`considered. Whereas, a cancer evasion from immune
`system is happened by several mechanisms like secreting
`factors,25
`reducing cytokine secretion,26
`angiogenic
`secretion.27
`surface antigen
`In
`this
`study,
`five
`immunotherapy approaches, alone and in combination
`with each other were applied to test groups and the
`outcomes were compared with those of the control
`group. Among test groups, BCG + HSP + Thalidomide
`demonstrated the most satisfactory results. Five out of
`the eight mice in this group survived and went into
`complete remission. Furthermore, the inhibition of the
`tumor size from 7th week was obvious in this group. The
`positive impacts seem to be due to individual and
`interactive features of the treatments. For instance, the
`chemical structure of the CWS of Mycobacterium bovis
`BCG strain
`(BCG-CW) has biological activities,
`especially immune-adjuvant and antitumor activities.
`Because of the availability and easy usage, it is
`considered as an immunotherapeutic tool.28 BCG also
`increases the expression of CD80, CD83, CD86, MHC,
`IL-12 as well as TNF-α secretion from the dendritic
`cells.11,12,29 On the other hand, heat shock proteins
`(HSPs) are employed to inhibit the aggregation of other
`damaged proteins and assist with
`refolding and
`reactivation of
`the damaged proteins under stress
`conditions.30 Several studies have investigated HSPs as
`tumor rejection antigens. In some cases, exogenous stress
`proteins seem to act as vehicles for the delivery of
`antigens to professional antigen-presenting cells (APC),
`consequently in cross-priming. It has been proved that
`HSPs can cooperate with various receptors on APCs
`resulting in HSP-peptide uptake, antigen cross priming,
`secretion of pro-inflammatory cytokines and maturation
`of the dendritic cells. Accordingly, the association of
`some stress proteins seems to induce both innate and
`
` 468 | Advanced Pharmaceutical Bulletin, 2014, 4(Suppl 1), 465-470
`
`adaptive immunity several folds.9 Some HSPs from
`tumor cells have been discovered to be able to start
`particular
`immunity
`against
`the
`tumor,
`through
`combination of the entire antigens and delivering them to
`APCs on MHC-I to activate tumor-antigen-specific CTL.
`The interaction of HSPs with APCs results in the
`secretion of pro-inflammatory cytokines like Interferon-α
`by dendritic cells (DCs) and macrophages.31,32 Moreover,
`presence of these molecules leads to decreased tumor
`size and increased survival rate.9 Angiogenic factor
`secretion from the tumor cells is the most important way
`to develop tumors in body and metastatic invasion to
`organs far from the primary tumor. Cancer cells by
`producing angiogenic factors such as VEGF, bFGF and
`TGF-β cause new vessels and expand the existing
`vessels.18 Thalidomide is one of the preventative factors
`that prohibit the formation of new vascular network from
`existing blood vessels. One of
`the goals of
`the
`angiogenesis inhibition is reducing blood flow to the
`tumor in order to limit nutrients and oxygen supplies for
`malignant cells.33
`these drugs could
`that each of
`Despite
`the fact
`individually result in anti-tumor effects, none of them
`was able to inhibit the tumor growth or improve the
`survival rate solely. In groups with only one treatment,
`since a few mechanisms of tumor repression were
`involved, anti-tumor impacts was not significant. In
`binary group BCG + Thalidomide, the average survival
`time of the mice was higher than individual groups and
`the mean size of tumors was lower than monotherapic
`and control groups. In the contrary, drugs in combination
`therapy especially with more than two drugs had the
`ability to inhibit the tumor development via different
`mechanisms.
`
`Conclusion
`In trilogy group BCG + HSP + Thalidomide, the anti-
`tumor effects were more than the other groups. This
`
`Genome Ex. 1048
`Page 4 of 6
`
`

`

`
`
`Effects of Some Immunomodulators on Survival and Tumor Size
`
`could be due to the synergistic effects of drugs and
`involvement
`of multiple
`inhibitory mechanisms
`including angiogenesis repression,
`improved
`tumor
`antigens
`presentation
`and
`the
`immune
`system
`fortification. Complementary studies to discover the
`mechanisms of action are encouraged.
`
`Acknowledgements
`We would like to thank for Hematology and Oncology
`Research Centers, Immunology Research Center (IRC)
`and Drug Applied Research Center especially Animal
`House for kind assistance. This work was supported by a
`grant from Hematology and Oncology Research Center.
`
`Conflict of interest
`The authors report no conflicts of interest.
`
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`2. Gupta S, Sussman DA, Doubeni CA, Anderson DS,
`Day L, Deshpande AR, et al. Challenges and possible
`solutions to colorectal cancer screening for the
`underserved. J Natl Cancer Inst 2014;106(4):dju032.
`3. Olszanski AJ. Current and future roles of targeted
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