The effect of Heat Shock Protein Inhibitors on cytotoxicity of Oxaliplatin in an in-Vitro model of

HIPEC in colorectal malignancies

ABSTRACT

In the management of peritoneal carcinomatosis, Hyperthermic intraperitoneal chemotherapy

(HIPEC) in combination with cytoreductive surgery is a promising treatment strategy. In our study
we studied the role of heated chemotherapy on heat shock protein (HSP) expression and induction
of cancer cell death and survival. Experiments were performed in one human colon cancer cell line
LoVo, isolated from supraclavicular lymph node metastasis and the murine colorectal cancer cell
line, CT26. In addition to their growth in monolayers, LoVo and CT26 were grown in a three
dimensional settings as spheroids. The antitumor efficacy of oxaliplatin and mitomycin C as a
single agent or in combination along with heat shock proteins inhibitors, HSP70 and HSP90 were
analysed, following incubation for 90minutes at either 37°C or 43°C. Cell viability was assessed
using a CellTiter-Glo chemiluminescence assay. Results displayed that LoVo cells were the most
sensitive cell line to hyperthermia. Administered as a single agent, oxaliplatin (1-20µM) showed
enhanced cytotoxicity in LoVo cells at 43°C compared to 37°C (p ≤ 0.05). Interestingly, CT26 cells
treated with oxaliplatin displayed increased cell viability under hyperthermic conditions compared
with those when treated at 37°C. Results remained the same when these cell lines were treated
dually with HSP inhibitors and oxaliplatin under hyperthermic conditions. Results observed for
LoVo and CT26 cultured in a monolayer were consistent with those seen in their respective 3D
models. The addition of mitomycin C to hyperthermia and oxaliplatin showed no synergistic
tumoricidal activity in any of the cell lines. The combination of Hyperthermia with oxaliplatin
alone and in combination with HSP inhibitors results in an increased tumoricidal activity in the
LoVo cell line. This in vitro model presents an opportunity to further investigate different heat
shock proteins and their inhibitors along with their mechanism, efficacy, and biological pathways.

INTRODUCTION

Peritoneal carcinomatosis (PC) secondary to colorectal cancer occur in approximately 10% of all
colorectal cancer patients. Colorectal cancer is the second most common cancer in women and third
most common cancer in men1. Up to twenty percent of colorectal cancer patients have metastatic
lesions at diagnosis and around fifty percent of patient develop metastasis during the duration of the
disease2. The most common sites of its metastases are the liver, lung and peritoneum 3 with
colorectal peritoneum metastases occurring in 20% of colorectal cancer patients 4. Colorectal cancer
has multiple risk factors among these diet is the most important exogenous risk factor in the
aetiology of colorectal cancer5. According to World Cancer Research Fund and the American
Institute for Cancer Research colorectal cancer can be prevented by appropriate diet and associated
factors5. Colorectal cancer may be diagnosed on the basis of symptoms presented by the patient or
on the basis of screening programmes. Screening is mostly done for detecting 90% of the sporadic
cases of colorectal cancers. Two types of screening are in used now-a-days, faecal occult blood test
(FOBT) and endoscopy. Biomarkers also play a role in early detection and diagnosis of this cancer.
Different types of proteins, glycoproteins, cellular and hormonal substances have been used to act
as a biomarkers but among them none has been found to be specific for colorectal cancer 6.
Carcinoembryonic antigen (CEA), Dukes criteria and several new carbohydrate antigen such as CA19-
9 are being used as its tumour markers.

The current treatment regimen consists of cytoreductive surgery combined with heated
intraperitoneal chemotherapy (HIPEC). HIPEC is a type of multimodal therapy for treating colorectal
cancers and other abdominal cancers. This loco regional therapy allows treatment of only specific
surfaces due to its minimal exposure to other parts of the body, as it remains confined within the
abdominal cavity. HIPEC is suitable for patients with advanced cancers within the abdomen, without
the involvement of disease outside the abdomen. HIPEC involves two phases, cytoreductive surgery
followed by heated chemotherapy7.

Cytoreductive surgery needs to be performed before HIPEC. This is done to remove all visible parts
of the tumour from abdominal organs, which in some cases, needs the whole organ to be removed
for achieving complete cytoreduction e.g. omentum, ovaries, colon, uterus, spleen 8. Following this,
heated sterilized chemotherapeutic solution is circulated through the abdominal cavity in order to
kill the remaining cancerous cells. Previously, chemotherapeutic drugs used to be given either orally
or in the form of intravenous injection to the patients, but now, HIPEC has replaced this, which is
performed at the temperature of 42-43°C for 30-60 minutes3, 7. The process consists of adding
chemotherapeutic drug to the carrier solution, which is already added to the abdominal cavity; this
is then circulated through the abdomen for 60-90minutes approximately 1, 3. When done, solution is
taken out of the abdominal cavity from the opposite side followed by closure of the incision (RR2).
The discussion relating to the intraperitoneal chemotherapeutic drug of choice is still unclear.
Although different drugs are being used during the HIPEC procedure which include Oxaliplatin,
Mitomycin C (MMC), Cisplatin and Irinotecan 1. Along with these drugs new study has also shown the
use of heat shock protein inhibitors as a combination therapy.

Oxaliplatin, a third generation platinum-based antineoplastic drug, works by forming DNA adducts
specifically in cancer cells, preventing DNA replication and transcription which ultimately causes
cancer cell death. Oxaliplatin undergoes non-enzymatic conversion to active derivatives through
displacement of the liable oxalate ligand. This di-amino-cyclo-hexane carrier ligand inhibits the
synthesis of DNA. Apoptosis may also contribute in the mechanism of action of oxaliplatin 8.
Mitomycin C is an antibiotic and antitumor drug which covalently binds DNA forming intrastrand and
interstrand crosslinks. It inhibits DNA synthesis and is also used in stem cell culture 9. Oxaliplatin has
now become a standard systemic treatment in treating colorectal cancers 10,11, while Mitomycin C is
only used as salvage treatment in advanced progressive colorectal cancer 12,13.

According to cancer research UK these cytotoxic drugs also have some side effects. Common side
effects of oxaliplatin present in more than 30% of the patients are; Peripheral sensory neuropathy,
and reversible acute, cold related dysesthesia. Unlike other platinum drugs oxaliplatin induces no
hepatic or renal toxicity. Common side effects of MMC present in approximately 10% of the patient
include; Increased risk of getting an infection, tiredness and weakness during and after treatment.
The use of MMC is limited because of its toxic effects. This drug induces cardiotoxicity,
nephrotoxicity and hepatotoxicity.

ADVANTAGES AND DISADVANTAGES OF HIPEC

Cytoreductive surgery and HIPEC have become an established mode of treatment in peritoneal
carcinomatosis varying from ovarian, colorectal, and gastric cancer, during the last decade 7. Along
with this it has many other benefits. For instance HIPEC is commonly administered as a single
treatment whereas standard chemotherapy requires several sessions. With HIPEC treatment, the
drug can be delivered in higher doses directly to the abdominal cavity, leading to a reduction in the
toxic effects associated with the drug 7. More importantly, because the abdomen is rinsed and all
leftover drugs are removed at the end of treatment there is reduced risk of systemic absorption,
meaning that any side effects that occur are of a much shorter duration 3,4. The constant
hyperthermia during HIPEC is thought to increase the penetration depth of chemotherapy into
tumour nodules.
Shortcomings of the HIPEC procedure have shown that the morbidity and mortality associated with
HIPEC were not significantly superior compared to those seen in response to other major
gastrointestinal interventions. Additionally as HIPEC doesn’t currently have standardised guidelines,
the drugs and their doses that are utilised in HIPEC vary among hospitals 6. Other significant
variations within the HIPCE procedure include the technique (open or closed abdomen), the
duration, the timing of drug deliver, and the volume of the perfusate, the kind of carrier solution, the
intra-abdominal temperature and the flow rate 2,7

HEAT SHOCK PROTEINS DURING HIPEC PROCEDURE

Heat shock proteins are a family of evolutionary conserved proteins induced by cellular stressors.
Five decades ago these proteins were discovered in the fruit fly Drosophila melanogaster 14.
According to some studies different HSP have been altered in different types of cancers. For example
HSP 70 is present in breast, prostate, hepatocellular cancers etc. while HSP90 is found in bladder
cancers, leukaemia and lung cancers14. Different cancers studies showed that there is an association
between HSP expression and multidrug resistance 15 as well as increased tumorigenesis -related
apoptosis16. Some researchers are of the view that the upregulated HSP cause increased resistance
against many anticancer drugs in cancer cells. While some studies shown that overexpression of HSP
eliminates the harmful effect of gamma radiation in tumour cells 17, 18, 19.

Previous studies have shown that cells could mount a specific transcriptional activity when exposed
to high temperatures that was described as heat shock response 14. This information leads to the
identification of HSP’s which influence many areas of science and medicine, and also helps in
understanding the pathology of many human diseases. Various expression of individual heat shock
proteins occurs in a wide range of cancerous processess 14, 18.

HSP’s play an important role in HIPEC. These proteins have been observed to be present on the
plasma membrane or the cell membrane of the cell and are found in both prokaryotic and eukaryotic
cells. These proteins act as molecular chaperones and protect cells from damage and apoptosis 20,21.
Their interaction with proteins of cell signalling pathways results in decreased apoptosis and
increased cell migration, proliferation and angiogenesis 21,22,23,24. Therefore, HSP are important players
in the development and progression of cancer and hence are now-a-days prime therapeutic
targets14.

The levels of these HSP are expressed in response to different environmental, physical and chemical
stress factors for example cytotoxic agents and hyperthermic conditions. Overexpression of these
proteins have been shown to be associated with poor prognosis with many types of cancers. Heat
shock protein 27, 70 and 90 (HSP27, HSP70 & HSP90) have been shown to be unusually
overexpressed in many types of cancers 14, 18. Recent studies showed that HSP’s are highly expressed
in HIPEC treated cancer cells resulting in resistance to hyperthermia- induced cancer cell death.
Therefore inhibition of HSP could potentially be promising strategy to improve the resistance of
colonic cancer cells and outcome of patients undergoing HIPEC treatment 25, 26.

AIMS

To investigate the effect of Heat shock protein inhibitors along with oxaliplatin and mitomycin C in
different colonic cancer cell lines.

This was achieved by

Measuring the effect of heated oxaliplatin and MMC in 2D and 3D.

Measuring the combined effect of oxaliplatin and MMC in 2D and 3D.

Measuring the combined effect of oxaliplatin and HSP inhibitors(HSP70&HSP90) in 2D and 3D.

MATERIALS AND METHODS

CELL CULTURE
The cell lines LoVo and CT26 were purchased from the American Type Culture Collection.
Oxaliplatin powder was purchased from Cambridge Biosciences (Cambridge, UK). All other
consumables unless stated were purchased form Sigma Aldrich, (Dorset, UK). LoVo and CT26
were cultured in Roswell Park Memorial Institute(RPMI) 1640 medium. This media was
supplemented with 10% heat inactivated fetal bovine serum and a 100units/ml penicillin G
and 100ug/ml streptomycin. Cell lines were cultured separately in 75cm 2 flasks and
passaged when approximately 80 to 90% confluent under aseptic conditions. A 1:10 split of
the cells was routinely used for continuity of the cell line in culture.
DETERMING CELL COUNT
For determination of the cell count cell/trypan mixture was transferred to a
haemocytometer by capillary action. Under light microscope (10 objective lens) the total
number of cells were counted in the central and corner four squares along with the top and
right-hand lines of each square.
PLATING OUT OF CELLS
After determining cell count, 2000- 5000cells/well and 1000cells/well were plated out in
96well plates for 2D and ultra-low attachment plates for 3D cell culture respectively. All the
cells were placed in the incubator for 24hours for 2D cell culture. For 3D they were given
more than five days to grow to an appropriate size.
DOSING OF CELLS
For dosing in 2D and 3D a fixed concentration of MMC, HSP 70 and HSP90 inhibitors were
added while the master stock solution of oxaliplatin (10mM) was diluted in an appropriate
volume of DMSO to make different sub stocks. These were then further diluted in the
appropriate media for application to cells in triplicate across a range of different
concentrations. The final concentration of DMSO per well was 0.3%. For negative control we
dosed 0.3% DMSO made up in media. Once cells were exposed to different concentrations
of oxaliplatin or controls, they were given heat treatment at particular temperature for
90minutes and then incubated in 37C incubator for a period of 72hours.
HEAT TREATMENT
For heating up cells in 2D and 3D we used only two temperatures 37°C and 43°C. The cells
were plated out in 96well plates and immediately subjected to the respective temperatures
in a temperature controlled incubator for 90minutes. Following heat treatment the drug
was replaced with fresh media and placed straight into the incubator. Cell viability was
assessed by ATP assay after 72hours in 2D and 3D.
DETERMINING CELL VIABILITY
After dosing of cells for the time period, 20ul of ATP solution (CellTiter GLO, Promega, and
Southampton, UK) was added to each well. Absorbance at a wavelength of 490nm, was
assessed using the VarioskanTM Flash Multimode Reader (ThermoFisher Scientific, Paisley,
UK) and cell viability expressed as a percentage of control cells.

STATISTICS
Graphs shown are expressed as the mean ± % SD (n=6 replicates). Sigmaplot 13.0 was used
to carry out statistical analyses. All data set were checked for normality via the Shapiro-Wilk
test. Data which passed normality testing underwent analysis by a two- way ANOVA, those
which did not underwent the non-parametric Holms-Sidak test. Significance was determined
from a p value < 0.05 shown as *.
RESULTS
Colorectal cancer lines were utilised for evaluating the cytotoxic effect of oxaliplatin in
conjunction with MMC, HSP inhibitors and heat. According to ATCC the tissue of origin for
LoVo and CT26 is the colon. LoVo is metastatic colonic cancer cell line while CT26 is a mouse
colonic cancer cell line.

A. LoVo B. CT-26

Figure 1 - The different colonic cell lines grown in 2d cell culture. Morphologically, (A) LoVo is
epithelial in nature while (B) CT-26 have a fibroblast morphology. Images were taken using a
Nikon digital camera head and its display unit.

EFFECT OF HEAT ON DIFFERENT COLONIC CANCER CELL LINES

To assess the effect of heat alone, both the cell lines were subjected to heat at 43C for a
duration of 90minutes.
 88

86

84

82
Cell viability (%)

80

78

76

74

72

70

68
CT26 LOVO

Figure 2 - A graph displaying the effect of heat on cell viability in 2D cultured colorectal
cancer cells. Data are presented as the average (n=5).
The results from figure show that LoVo cells are much more sensitive to the cytotoxic effects
of heat when compared to CT26 cell line.

CYTOTOXICITY OF OXALIPLATIN IN CONJUNCTION WITH HEAT TREATMENT OVER 72HRS IN

2D
Subsequently both the cell lines were grown in 96well plates and then subjected to various
concentration of oxaliplatin. Immediately after dosing, the cells were given heat treatment
at 37°C and 43°C for 90minutes, after which they were washed and replaced with fresh
media and then incubated at 37°C for 72hrs.

[A] [B]
 Figure 3 – Graphs displaying the effect of immediate heat treatment on the sensitivity to
oxalipaltin. (A)LoVo, (B) CT26 cells in 2D were dosed with various concentrations, of oxaliplatin
immediately followed by heat treatment at 43°C for 90minutes. Cell viability was determined by
an ATP assay 72hours after heat treatmnet. Data are shown as the average, where error bars
repersent +_%SD (n=6). Cell survival for each temperature was corrected for heat cytotoxicity.
Statistically significant differences are highlighted using * (p ≤ 0.05).

Oxaliplatin displayed enhanced cytotoxicity in LoVo cells when they were subjected to heat
as shown in figure3A. CT26 on the other hand showed increased cell viability at 43°C
compared to 37°C as shown in figure 3B.
CYTOTOXICITY OF OXALIPLATIN AND MITOMYCIN C IN CONJUNCTION WITH HEAT
TREATMNET OVER 72HRS IN 2D
LoVo and CT26 were subjected to different concentration of oxaliplatin and fixed
concentration of MMC. Right after treatment, both the cell lines were subjected to heat
treatment at 37°C and 43°C for a duration of 90minutes. After heat treatment both the cell
lines were washed and replaced with fresh media after which these were incubated at 37°C
for 72hrs.

[A] [B]
Figure 4 –Graphs displaying the effect of immediate heat treatment on the sensitivity to
oxaliplatin in combination with MMC. (A)LoVo, (B) CT26 cells in 2D were dosed with various
concentrations of oxaliplatin together with MMC at ..uM, followed by heat treatment at 43°C for
90minutes. Cell viability was determined by an ATP assay 72hours after heat treatment. Data are
shown as the average, where error bars represent ±%SD (n=6). Cell survival for each
temperature was corrected for heat cytotoxicity. Statistically significant differences are
highlighted using * (p ≤ 0.05).

LoVo and CT26 did not show any decrease in cell viability even after treatment with
oxaliplatin and MMC. The cell viability remained same at 37°C and 43°C in both the cell
lines.
CYTOTOXICITY OF HEAT SHOCK PROTEIN INHIBITORS IN CONJUNCTION WITH HEAT
TREATMENT OVER 72HRS IN 2D
Heat shock proteins inhibitors in conjunction with heat treatment were measured following
72hrs incubation at 37°C.
[A] [B]

[C] [D]

Figure 5 – Graphs displaying the effect of immediate heat treatment on the sensitivity to
HSP inhibitors.(A) LoVo with 17-AAG,(B) CT26 with 17-AAG,(C) LoVo with VER155008,(D)
CT26 with VER155008. These cells were dosed with various HSP inhibitors concentrations
immediately followed by heat treatment at 43°C for 90minutes. Cell viability was
determined by an ATP assay 72hours after heat treatment. Data are shown as the
average, where error bars represent ± %SD (n=6). Cell survival for each temperature was
corrected for heat cytotoxicity. Statistically significant differences are highlighted using *
(p ≤ 0.05).

Both the cell lines when treated separately with HSP inhibitors (HSP70 and HSP90) showed
no significant changes in cell viability. It can be seen from the figure that heat treatment did
not potentiate the cytotoxicity of the HSP inhibitors.

CYTOTXICITY OF OXALIPLATIN IN CONJUNCTION WITH HEAT SHOCK PROTEINS INHIBITORS

AND HEAT TREATMENT OVER 72HRS IN 2D
Both the cell lines were subjected to different concentration of oxaliplatin with fixed
concentration of HSP70 inhibitor (VER155008) and HSP90 inhibitor (17-AAG) in conjunction
with heat treatment.

[A] [B]
Figure 6 –Graphs displaying the effect of immediate heat treatment on the sensitivity to
oxaliplatin in combination with HSP inhibitors. (A) LoVo (B) CT26 cells in 2D were dosed
with various concentrations of oxaliplatin together with HSP inhibitors at 1uM, followed
by heat treatment at 43°C for 90minutes. Cell viability was determined by an ATP assay
72hours after heat treatment. Data are shown as the average, where error bars represent
± %SD (n=6). Cell survival foe each temperature was corrected for heat cytotoxicity.
Statistically significant differences are highlighted using * (p ≤ 0.05).

Oxaliplatin in combination with HSP inhibitors displayed enhanced cytotoxicity in LoVo cells
when subjected to heat treatment as shown in figure6A. CT26 on the other hand showed
increased cell viability as shown in figure 6B.
GROWTH OF SPHEROIDS:
Spheroids for both the cell lines were grown for 5 days varied in shape and size from their
day 1 to day 5, as shown in Figure 7A. The outer margin of the day 1 spheroids were
irregular and its centre was not clearly defined while on day 5 the fully grown spheroid
showed well demonstrated outer margins. By Day 5 the majority of spheroids had an
appropriate diameter, hence they could be used for dosing.
Figure 7 - Images of representative CT26 spheroids cultured from 1000 cells on ULA plates over 11 days. Treatment was initiated on Day 5.
Images are taken from untreated spheroids. (Images were captured using a Nikon digital camera head and its display unit. Scale bar = 100 μm.
Figure 8- Images of representative treated or control LoVo spheroids cultured from 1000 cells on ULA plates over 8 days. Treatment was
initiated on Day 5. (A) Images are taken from spheroids treated at 37°C. (B) Images are taken from spheroids treated at 43°C. (Images were
captured using a Nikon digital camera head and its display unit. Scale bar = 100 μm.
CYTOTOXICITY OF OXALAIPLTIN IN CONJUNCTION WITH HEAT SHOCK PROTEINS
INHIBITORS AND HEAT TREATMENT OVER 72HRS IN 3D

LoVo and CT26 cells were dosed with different concentrations of oxaliplatin and fixed
concentration of HSP70 and HSP90 inhibitors in 3D. After dosing with different drugs
concentrations, the cell were immediately subjected to heat treatment at 37°C and 43°C for
90minutes. For assessing cell viability, ATP assay was performed after 72hrs.

[A] [B]

Figure 9 – Graphs displaying the effect of immediate heat treatment on the sensitivity of
oxaliplatin and HSP inhibitors. (A) LoVo cells (B) CT26 cells in 3D were subjected dosed
with various concentrations, immediately followed by heat treatment at 43°C for 90
minutes. Cell viability was assessed by an ATP assay 72hours after heat treatment. Data
are shown as the average, where error bars represent +_ %SD (n=6).

DISCUSSION
Currently cytoreductive surgery with HIPEC represents a promising therapeutic strategy in
many peritoneal cancers. Many researchers working on cancer therapy are focussed on how
to suppress the levels of HSP as these are well known to be overexpressed in many types of
cancers. Overexpressed HSP results in inhibition of cell apoptosis and increased resistance
to the chemotherapeutic drugs. Thus, the inhibition of HSP has become an important
strategy in cancer therapy27. In present study, an in vitro approach was applied to evaluate
the cytotoxic effects of oxaliplatin alone or in combination with MMC, HSP inhibitors,
together with heat in two colorectal cancer cell lines. Previous experimental studies have
shown beneficial effects by combining heat with chemotherapeutic drugs 25,28. It is thought
that localized heating may lead to an enhancement in drug cytotoxicity, i.e. improved
efficacy, within a defined target region. Here in addition to 2D cell culture, spheroids were
applied as an additional model system, since they have been shown to be
pathophysiological similar to tumour cells. This is due to their ability to replicate the
microenvironment as well as displaying cell interactions that would conventionally not be
seen in traditional 2D cell culture system. Different signalling pathways, protein induction,
inhibition, and phosphorylation. For example the Mitogen- activated protein kinase (MAPK)
pathway, AKT-Mtor-S6k pathway or ERK phosphorylation act in a similar manner in 3D as in
their natural environment but different in 2D model system.
We hypothesised that heat increases the permeability of cancer cells and results in killing
large proportion of colonic cancer cell lines. A number of previous and recent studies
showed that hyperthermia increased the tissue absorption of chemotherapeutic drugs and
hyperthermia along with chemotherapeutic drugs exert beneficial results in many settings 27.
Alongside this, various experimental studies have shown the beneficial effects of using
Oxaliplatin, MMC and HSP inhibitors together with heat 27. Research work from certain
studies showed that oxaliplatin efficacy can be enhanced with heat 27. In these studies
temperatures between 38-43°C were seen to be appropriate for testing the efficacy of
HIPEC in colorectal cancers29.
For visualising the effect of heat alone, different cell lines were studied with the only
variable being temperature. All the cell lines were treated with Oxaliplatin, MMC and HSP
inhibitors alone as well as in combination. Results showed that LoVo cells were more
sensitive than other cell lines, which can be most likely aligned to the cytotoxic effect of
heat itself. This would have led to protein denaturation meaning a decrease in DNA repair
processes and protein synthesis, which ultimately would cause cell death or an
enhancement in apoptosis. Similar to other data heat has been shown to play an important
role in apoptosis via activation of p53, Bax and the repression of Bcl-2 specifically in LoVo
cells30. While the CT26 appeared to be less sensitive may be due to an upregulation of HSPs
and other heat induced events resulting in the development of thermo-tolerance which
protects cells from stresses. Furthermore, heat has been shown to effect the perfusion and
oxygenation in normal and cancerous cells. Heat alone could increase the perfusion and
oxygenation in normal tissue while opposite could be the case in tumour cells leading to
hypoxia in cancerous cells27. In addition to this, functional proteins within the cancer cells
becomes unfolded, and the cell membrane, nuclear DNA, cytoskeleton, and cell organelle
can be damaged27. Heat shock proteins are thought to play an important role in this case as
these are induced in response to various physical, chemical and environmental stress factors
such as cytotoxic agents and hyperthermic conditions. These defensive proteins protect the
cells from apoptosis inducing events and play a significant role as cell chaperones and
protect cells from damage thus resulting in increased tumour cell viability. Here, we have
demonstrated the role of HSP inhibitors along with other cytotoxic drugs in colonic cancer
cell lines. HSP70 and HSP90 inhibitors have been used together as the dual targeting of both
the HSP70 and HSP90 might be effective in successfully inhibiting the HSP dependent
antiapoptotic pathways31. HSP 70 is responsible for blocking the extrinsic pathway of
apoptosis by interacting with ASK (apoptosis signal- regulating kinase) and JNK(c-Jun N-
terminal kinase (Upregulated art 38) while HSP90 support cell from apoptosis by directly
interacting with Apaf-1 and inhibiting the formation of a functional apoptosome and
recruitment and activation of procaspase932.
These findings indicate that heat treatment displayed significant synergistic interaction with
oxaliplatin monotherapy at 43±C only in the LoVo cell line. While the CT26 on the other
hand displayed that heat could not efficiently increase the chemotherapeutic effect of
oxaliplatin. A significant decrease in cell viability was observed in LoVo cell line, which can
be clinically significant to some extent, while the combination of oxaliplatin, MMC and HSP
inhibitors showed only 20-30% decrease in cell viability in CT26. When heat treatment was
combined with a fixed concentration of either MMC or HSP inhibitors, the results for the cell
lines were comparable with that induced by untreated cells. Here again, the spheroids for
LoVo were found to be less resistant as compared to CT26, which were found to be less
sensitive when compared to cells in 2D monolayer. This could be due to the
microenvironments present such as hypoxia and regions of proliferation. In addition to this
increased chemo-resistance could points towards the under/over expression of heat shock
proteins in 3D when compared to 2D cell culture. Furthermore heterogeneity in spheroids
could be one of the determinants of drug resistant. Several cellular microenvironment
conditions like necrosis and proliferation are responsible for its phenotypic heterogeneity.
LoVo cell appeared to be more sensitive as compared to CT26 cell line. The sensitivity of
LoVo may be due to its metastatic nature, as it initially found as a fragment of a metastatic
tumour nodule in the left supraclavicular region. Other factors to be considered include the
expression of certain proteins such as HSP27, HSP70, HSP90 etc. in 2Dvs 3D. The other
reason for its sensitive nature could be due to its expression for carcinoembryonic antigen
(CEA) while CT26 express mainly for beta galactosidase (beta-gal). Interestingly, our present
data demonstrated that the decrease in colonic cancer cell viability is greater in LoVo cells at
43°C when compared to at 37°C.
In conclusion, heat treatment combined with exclusive treatment with Oxaliplatin and HSP
inhibitors displayed a temperature dependent enhancement of cytotoxicity in LoVo cells.
However, no synergistic activities were seen when heat was applied to CT26 cells exposed to
Oxaliplatin in combination with either MMC or HSP inhibitors.