Lithuanian University of Health Sciences Department of General Surgery “`HIPEC´- Hyperthermic Intraperitoneal Chemotherapy. Evaluation of Intraperitoneal free cancer cells.” Lucía Romero Ramil Antanas Gulbinas Vaidotas Cesna Kaunas 2016/2018

Lithuanian University of Health Sciences

Department of General Surgery

“`HIPEC´- Hyperthermic Intraperitoneal Chemotherapy. Evaluation of

Intraperitoneal free cancer cells.”

Lucía Romero Ramil

Antanas Gulbinas

Vaidotas Cesna

Kaunas

2016/2018

TABLE OF CONTENTS

SUMMARY ………....………… 3

ACKNOWLEDGEMENTS ……… 4

CONFLICT OF INTEREST ………... 5

CLEARANCE ISSUED BY ETHICS COMMITTEE ………...…… 6

ABBREVIATIONS ……… 7

INTRODUCTION ………. 8

AIM AND OBJECTIVES OF THE THESIS ……… 9

LITERATURE REVIEW ………. 10

MATERIALS AND METHODS ……….. 11

RESULTS ………. 13

DISCUSSION OF THE RESULTS ……….… 14

CONCLUSIONS ………. 15

FIGURES ………...……… 17

SUMMARY

Lucía Romero Ramil. “HIPEC” Hyperthermic Intraperitoneal Chemotherapy. Evaluation of Intraperitoneal free cancer cells.

HIPEC is a great treatment option to cure GI and ovarian cancers at advanced stages. Also it improves survival rates. However there is a lack of knowledge about this method.

The aim of this work is to evaluate energetic changes of intraperitoneal free cancer cells mimicking HIPEC conditions.

Objectives: To evaluate cell viability. To evaluate cell mitochondrial respiratory rate and to evaluate ATP changes in cells affected by temperature and chemotherapy.

Methods: cell viability was measured using MTT assay while Oroboros O2k-FluoRespirometer was used to control mitochondria’s respiratory function.

Conclusions: changes in mitochondria´s function were show for AGS cell line, but there is a lack of research to compare results.

ACKNOWLEDGEMENTS

This research is a part of my integrated studies in the Lithuanian University of Health Sciences as a Final Master Thesis, during my 6th year of medical studies.

I would like to thank personally Vaidotas Cesna and also Professor Antanas Gulbinas who helped, taught and advised me throughout the whole research work.

CONFLICT OF INTEREST

CLEARANCE ISSUED BY ETHICS COMMITTEE

ABBREVIATIONS

CO2- Carbon Dioxide

CRS- Citoreductive Surgery

CI- Combination Index

CI- Complex I

CII- Complex II

DI- Deionized

DMSO- Dimethylsulfoxide

E.g.- Example given

GI- Gastrointestinal

HIPEC- Hyperthermic Intraperitoneal Chemotherapy

ICP-MS - Inductively Coupled Plasma Mass Spectrometry

IC- Inhibitory Concentration

IFCC- Intraperitoneal free cancer cells

IC 50- Half maximal inhibitory concentration

MEE- Median-Effect Equation

MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OXPHOS-oxidative phosphorylation

PSMs- Peritoneal Surface Malignancies

PM- Peritoneal Metastases

RPMI-1640- Roswell Park Memorial Institute Medium

RCI- respiratory control index

INTRODUCTION

Gastric cancer is one of the leading causes of cancer in the world. It is hard to diagnose and most of the time diagnosis is made too late, when cancer has already spread.[1] The main route of spreading of gastric cancer is through the peritoneum and these metastases are called peritoneal carcinomatosis (PC).[2] Because of its way of dissemination, it is related to poor prognosis and poor survival rates.

One of PC treatment modalities is, after an optimal citoreductive surgery (CRS), the combination treatment of hyperthermia and chemotherapy, also called HIPEC (hyperthermic intraperitoneal chemotherapy).[3] The main idea of the combination therapy is to access a higher number of cancer cells by delivering intraperitoneally heated chemotherapy.[4] Hyperthermia is believed to contribute in this process, by enhancing the cytotoxic effect of chemotherapy.[5] Many articles establish HIPEC, as the only treatment modality that has shown to increase 5-year survival rates and that has been able to reduce recurrence of PCs.[5]

Although the synergistic effect of hyperthermia on chemotherapy has been studied in a large number of occasions, there is not enough information about how hyperthermia affects cellular energetics.[6] Thus, the research of mitochondrial function should be a crucial step, since it plays an important part in cell mediating processes. Mitochondrion plays a multi-factorial roll in the cell: it produces energy by synthesizing ATP, it is responsible for apoptosis, detoxification and Ca2+ buffering among others.[7] Despite the fact that modifications in the mtDNA of cancer cells are not a recent finding, attention was drawn to this mtDNA variations when the mitochondrial tricarboxylic acid (TCA) cycle gene mutations were found.[8] Since distinct cancer cells undergo distinct bioenergetic changes; some tend more to glycolysis others tend more to oxidative phosphorylation, the reaction of cancer cells to combination therapy at a mitochondrial level, should be evaluated in detail.[9]

The aim of this study is to contrast the effect of thermochemotherapy (40 - 43ºC) on mitochondrial oxidative phosphorylation and to assay whether hyperthermia has a synergistic effect on cisplatin in AGS cells.

AIM AND OBJECTIVES

The aim of this work is to evaluate energetic changes of intraperitoneal free cancer cells mimicking HIPEC conditions.

Objectives: To evaluate cell viability. To evaluate cell mitochondrial respiratory rate and to evaluate ATP changes in cells affected by temperature and chemotherapy.

LITERATURE REVIEW

Chemotherapy, when administered intraperitoneally, results in a positive flow gradient, which is maintained by peritoneal barrier. Peritoneal barrier does this by keeping a low concentration in plasma vs. a high one intraperitoneally.[10] Hyperthermia enhances the effect of chemotherapy by its two cytotoxic effects: primary effect (protein denaturation, impairment of the DNA repair…) and secondary effect (uptake by tumor cells is increased; it potentiates the action of some drugs…). After an optimal CRS, two catheters are placed in the abdominal cavity. One an inflow catheter and the other an outflow catheter. Succeeding the introduction of the catheters, the abdominal cavity is filled with a normal saline solution. Connecting both catheters to a roller pump and a heat exchanger hyperthermia of the solution is obtained. The effective range of penetration is 1-3mm. There are two main techniques in which this is accomplished, the `open´ technique and the `closed´ technique. Duration of HIPEC varies between centers, but ranges between 30min and 2 hours.[11] It can be administered same time as surgery or right after it.[12] Certain features must be met, among drugs, for a proper absorption of chemotherapy into systemic circulation. Molecular weight and charge, hydrophilicity and drug heat stability should be confirmed. Noting that the heat has limited penetration, hyperthermia during HIPEC ranges from 41-43ºC. Patients fitting the criteria to undergo HIPEC treatment, include patients with PC mainly arising from: colorectal, gastric and ovarian cancers.[13] The transmission of hyperthermic chemotherapy into the peritoneum was firstly explored by Koga et al. in 1988.[14] There are many articles and data that agree (based on scientific analysis) on the benefits of the combination of CRS and HIPEC.[15] Harmful effects of HIPEC when combined with CRS are mainly related to the movement of the therapeutic agent into the systemic circulation.

MATERIALS AND METHODS

For this experiment we used one human cell line, the AGS (gastric adenocarcinoma). It was acquired from the American Type Cell Culture (ATCC Manassas, VA, United States).

Layout of the study

Cells were cultured using a RPMI-1640 medium (Gibco®), supplemented with 10% fetal bovine serum and a solution of penicillin (100 units/ml) + streptomycin (100 μg/ml). Cell flasks were cultured in a humid CO2 incubator, maintained in a 5% carbon dioxide environment with a temperature of 37ºC. During previously described conditions, cells were cultivated during 24 hours. Subsequently cells were treated with temperature or cisplatin and temperature plus cisplatin. Temperature levels ranged from 37ºC to 45ºC and doses of cisplatin IC50 were optimized for each cell line in the experimental setting. Extent of treatment was 60 minutes, in order to mimic real HIPEC conditions. Cells were warmed in CO2 incubators at particular Tº plans. When the temperature attained the desired level, one hour count back began. Consecutively after the treatment, the medium was replaced, and then cells were cultivated for another two days. Thereafter, viability assay and flow cytometry were executed.

Viability assay

Direct cytotoxic effects of cisplatin plus/or hyperthermia were determined using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay. 10.000 cells were plated in a 96-well plate. 5mg/mL of MTT reagent was aggregated to the well and the plate was incubated for 4 hours at room temperature, until intracellular purple formazan crystals were visible under microscope. The MTT reagent was removed and formazan crystals are then dissolved using DMSO (dimethylsulfoxide). The absorbance of the formazan crystals was determined spectrophotometrically at 570nm.

Apoptosis assay

After treatment with cisplatin IC50 and hyperthermia (43ºC), early apoptosis was assessed via

the Guava NexinTM assay. Cells cultivated with no cisplatin and 37ºC temperature were denominated as control cases. All the experiment steps were performed according the manufacturer’s instructions. Samples were evaluated using Guava PCA system and CytoSoft software.

Drug combination analysis by isobolograms

MEE (Median-Effect Equation) was used to give academic basis for CI (Combination Index) that concede measurable assessment of single drug effect, determining C<1 as synergism, C=1 summation and C>1 antagonism.[16] To establish the CI for the summation (C=1) action of cisplatin plus hyperthermia, a special form of the MEE equation, the isobol equation or isobologram, was used. Isobolograms were assembled within seconds with the third-generation software, CompuSyn. Basis for the isobologram analysis was gathered from the MTT assay.[17, 18] Temperature parameters fluctuated from 38ºC to 45ºC and cisplatin concentrations from 25, 50, 100, 200 to 400 μg/ml respectively. The combination of hyperthermia plus cisplatin data was the following: 39-44ºC + 50-200 μg/ml (E.g.: 39ºC+50 μg/ml; 39ºC+100 μg/ml and 39ºC+50-200 μg/ml; same combination for remaining temperature and doses).

Role of mitochondria

To control mitochondria’s respiratory function, we used the Oroboros O2k-FluoRespirometer powered with Datlab 5 software. Additional oxidative phosphorylation analysis was made by the isolation of mitochondria. Mitochondria were cultured using a mitochondrial respiration medium or MiR06. MiR06 contained the following: EGTA 0.5 mM, MgCl2 3 mM, Lactobionic acid 60 mM, Taurine 20 mM, KH2PO4 10 mM, HEPES 20 mM and D-Sucrose 110 mM pH was adjusted to 7.1 at 37ªC. For plasma permeabilization, Digitonin 1 µg/ml was added. State 2 respiration rate (Routine respiration) (V0) was evaluated by the addition of Complex I (CI) substrates: glutamate and malate

mitochondrial permeability. The ratio between State 2: State 4 also called, respiratory control index (RCI) shows the coupling between respiration and phosphorylation and it was calculated as VADP/VCAT. Whilst VADP+cyt c /VADP ratio was calculated as cytochrome c effect. Oxygen flux was associated to cell number as (pmol/s/1 mln cells).

RESULTS

Response to hyperthermia

Tetrazolium reduction assay determined direct cytotoxic effect of hyperthermia on gastric cells. AGS cell line was subjected to temperatures starting at 37ºC and ending at 45ºC (increasing 1ªC at a time). Settled the control case as: normothermia (37ºC) and no cisplatin. AGS cells were very susceptible to hyperthermia. From 38ºC - 41ºC viability remained constant but as hyperthermia was being reached viability dropped by 30% (at 43ºC 13%, at 44ºC 22% and at 45ºC 30%). AGS cells responded to hyperthermia as meant to, by gradually inhibiting cell viability. (Fig.1.).

Sensitivity of cisplatin

Cisplatin generated a suppressive effect of cell viability. AGS cells showed a sensitivity of cisplatin IC50 at 182 μg/ml. (Fig.2.) Inadequate temperatures can be harmful during association of

hyperthermia plus cisplatin. Cisplatin IC50 doses were previously determined for AGS cell line. Throughout the experiment, temperatures below 41ºC had no summation effect (C=1). But curiously, cell viability raised by 33% at 42ºC. On the other hand, thermochemotherapy decreased cell viability drastically, at 45ºC by 70%. (Fig.3.). Briefly, for AGS cells, combination therapy may magnify the cytotoxic effect of cisplatin while non-hyperthermic plans can even boost cell growing and aggravate presumed HIPEC effects.

Isobol equations: random response to combination therapy

Cisplatin plus hyperthermia effect assessment was made via isobolograms. Isobolograms gave a quantitative assessment of this combination therapy, being: CI<1 synergism, CI=1 summation and CI>1 antagonism. Association of hyperthermia plus cisplatin in AGS cells, with every combination of temperature and doses, showed CI>1 or antagonistic effect. (Fig.4.).

Effect on early apoptosis, when hyperthermia is added to cisplatin.

Early apoptosis was estimated via the ICP-MS assay. ICP-MS assay showed that early apoptosis was enhanced in cisplatin + hyperthermia- treated cells (raise of 61% on early apoptosis); while isolated cisplatin treatment did not significantly cause early apoptosis. (Fig. 5.).

Influence of cisplatin in AGS mitochondrial function

Effect of cisplatin on AGS bioenergetics was evaluated. We evaluated mitochondrial CI (Glut/Mal) and CII (succinate) dependent respirations (V0), active respiration (VADP), the coupling between respiration and phosphorylation or RCI (VADP/VCAT), the permeability of the inner (VADP+cyt c) and the effect of cytochrome c (VADP+cyt c /VADP).

With Glut/Mal as substrates data was the following. In AGS cells, cisplatin increased V0 by 2.44 fold and VADP by 34%. Whereas RCI (VADP/VCAT) tend to decrease by 44% compared to untreated cells (p<0.05).

With succinate as substrate, cisplatin increased VADP by 37% and VADP by 1.94 folds but it decreased RCI (VADP/VCAT) by 21%. Cytochrome c effect was incremented following the treatment with cisplatin 1.11 ±0.10 vs. control 1.03±0.04 in AGS cells.

Influence of hyperthermia in AGS mitochondrial function

Hyperthermia (40 – 43ºC), in AGS cell line, had effect in both State 2 (V0) and State 3 respiration (VADP) respiration rates. V0 respiration rate, at 40ºC, was incremented by 1.32, and by 1.64 fold at 43ºC. VADP respiration rate, as well, was increased by 14 and 30% (at 40 and 43ºC respectively). Consequently, RCI (VADP/VCAT) tent to decrease, at 40ºC, 16% and at 43ºC, 27%. Permeability of mitochondria´s inner membrane was increased, reflecting on a VCAT (with addiction of succinate as substrate) increment of 1.11 (at 40ºC) and 1.32 (at 43ºC). Nevertheless, outer membrane´s permeability was not affected by hyperthermia in AGS cells, showing no cytochrome c effect (VADP+cyt c /VADP). All compared to untreated cells (p<0.05), at 37ºC.

Mitochondria´s V0, VADP and RCI stayed unaltered throughout thermochemotherapy compared to untreated cells (at 43ºC with no cisplatin). (Fig.6.).

DISCUSSION

So far, knowledge regarding the additive effect of hyperthermia to chemotherapy is insufficient and uncertain.[19] In this in vitro experiment, we tried to determine how hyperthermia (40, 43ºC) affects cisplatin-treated AGS cells. Thermochemotherapy was administered for 1 hour, for the purpose of simulating real HIPEC conditions. Through SUIT protocol, we evaluated mitochondrial CI (Glut/Mal) and CII (succinate) dependent respirations, active respiration, the coupling between respiration and phosphorylation, the permeability of the inner and outer mitochondrial membranes and the effect of cytochrome c. First read-out was made at 48h since initial readouts did not prove any changes.

We selected one of the most common peritoneal invading cancers, that being AGS or gastric adenocarcinoma. This is a clinical review that aims to provide with a better understanding of gastric cancer cell response to combination therapy of cisplatin plus hyperthermia.

Notwithstanding the fact that cisplatin has been universally used in chemotherapy, cisplatin effects on a particular mitochondrial level have not being enough described.

Main conclusions taken from this study are that cisplatin damages mitochondrial function, by lowering OXPHOS and increasing inner membrane permeability. Hyperthermia (40-43ºC) damages mitochondrial function, and it does so by the uncoupling of OXPHOS. On the contrary, hyperthermia does not amplify cisplatin effect in AGS cell line. Mitochondria, as a consequence of loosing its membrane integrity, tend to decrease RCI.

Comparatively to our study, Custódio et al. investigated the effects of Cisplatin in rat liver mitochondria. Mitochondria were treated with 20 nmol/mg of cisplatin, which induced Ca2+ dependent mitochondrial swelling. To prevent this swelling, CyA was added. With higher cisplatin concentrations (40 nmol/mg) and using succinate as the substrate, State 2 and 4 of respiration were stimulated while State 3 of respiration was reduced. RCR, ADP/O and the ADP phosphorylation rates were also diminished. Authors end, determining that cisplatin interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to H+.[20]

Other researchers have also studied effects of cisplatin on mitochondrial function in cancer cells. Kirk A. Tacka et al. investigated the effects of cisplatin on Jurkat cells. They showed that doses of cisplatin of 0−25μM, for 3 h, had no instant effect on cellular mitochondrial oxygen consumption, but 24 h after exposure to cisplatin, respiration rates and cell viability decreased. They also exhibited the lack of effect on beef heart mitochondria under 0−25μM cisplatin, proposing that cisplatin may

attack mitochondrial respiratory chain indirectly. Authors conclude that Jurkat cell modifications in mitochondrial function are a secondary effect of cisplatin cytotoxicity.[21]

Anyhow, it is difficult to contrast results between studies since each cell line has shown to have a specific sensitivity to cisplatin. In our study, AGS cells showed IC50 of cisplatin of 182 μg/ml.

Mitochondrial cytochrome c (cyt c) has been identified as one of the essential molecules of apoptosis; it is also responsible for cellular metabolism.[22] In our study we saw a positive effect of cisplatin on cytochrome c; cytochrome c effect, was incremented following the treatment with cisplatin (1.11 ±0.10 vs. control 1.03±0.04) in AGS cells. By increasing inner mitochondrial membrane permeability cytochrome c is released, thus, inducing apoptosis.

Furthermore, hyperthermic (40-43ºC) effect on cisplatin vs. untreated cells (37ºC, no cisplatin) was examined. As said previously, AGS cells showed sensitivity for cisplatin at 182 μg/ml.

In AGS cells, hyperthermia induced uncoupling of OXPHOS thus decreasing RCI. Zukiene et al. proved that non-hyperthermic temperatures did not affect State 2. But as temperatures raised State 2 raised with them, showing the highest level at 45ºC, with a 5fold increase compared to control case, suggesting a strong uncoupling of OXPHOS due to raise in membrane leak.[23] Jarmuszkiewicz et al. studied the effect of hyperthermia (42ºC) on rat skeletal muscle mitochondria and revealed that hyperthermia caused a decrease in membrane potential, OXPHOS and H2O2 production.[24]

Hyperthermia is well known for enhancing cytotoxic effect of cisplatin, it does so by protein denaturation and impairment of the DNA repair among others. Our results showed a positive effect of hyperthermia, affecting mitochondrion of AGS cells.

It is highly recognized the synergistic effect that has hyperthermia to cisplatin. It is one of the very little treatment modalities that has shown to increase the overall 5-year survival rate and which decrease the extent of peritoneal invasion.[25] When we measured mitochondrial function of AGS cells, hyperthermia did not increment cytotoxic effects of cisplatin. Slight covering of the topic leads us with no options to compare our results to others. We also found a slight raise in cell viability of 16% in gastric cells. But on the other hand, thermochemotherapy had the opposite effect, thus decreasing cell viability up to 18%.

FIGURES

Fig.1. Effect of hyperthermia on cancer cell viability

Effect of hyperthermia on cancer cells viability. AGS cells were subjected to temperatures ranging

from 37ºC to 45ºC (raising 1ºC at a time), for 1 hour, and cell viability was measured. From 43ºC, viability gradually decreases for AGS cells. Data was compared to the control case (37ºC and no cisplatin; viability was set as 100%). Statistical analysis was performed using Mann Whitney test, comparing selected groups to control. * p<0.05.

C e ll v ia b il it y , % 3 7 3 8 3 9 4 0 4 1 4 2 4 3 4 4 4 5 0 2 5 5 0 7 5 1 0 0 T e m p e r a t u r e , oC * * _*

Fig.2. Effect of cisplatin on cell viability.

Effect of cisplatin on cell viability. Cisplatin generated a suppressive effect of cell viability. AGS cells

showed a sensitivity of cisplatin IC50 at 182 μg/ml. Data was compared to the control case (37ºC and

no cisplatin; viability was set as 100%). Data showed as mean ± standard error from ≥3 replicates

Fig.3. Effect of combination therapy on cell viability.

Effect of combination therapy on cell viability. Combination therapy of cisplatin and hyperthermia

had no significant effect on cell viability. Peaks of viability were shown. At 42ºC, viability raised by

uM C e ll v ia b il it y , % 0 2 5 5 0 1 0 0 2 0 0 4 0 0 0 5 0 1 0 0 A G S IC 5 0 T e m p e r a t u r e , oC C e ll v ia b il it y , % 3 7 3 8 3 9 4 0 4 1 4 2 4 3 4 4 4 5 0 2 0 4 0 6 0 8 0 5 0

Fig.4. Isobolograms and cell viability.

a.(I) b.(CIP)

Isobolograms and cell viability. A. Isobolograms. B. Combination Index Points. To establish the CI

(Combination Index) for the summation (C=1) action between cisplatin (A) and hyperthermia (B) isobols were used. Isobols exemplify conditions to inhibit cell growth by 50%. CI=1 (summation) is showed in the diagram as the straight line; C<1 (synergism) is shown as combination points below the CI line whereas C>1 (antagonism) is shown as combination points above the CI line. Association of hyperthermia plus cisplatin in AGS cells, with every combination of temperature and doses, showed CI>1 or antagonistic effect.

Fig.5. Cisplatin and temperature. Effect on apoptosis.

(A)

(B)

37ºC-cis 37ºC+cis

43ºC+cis

Cisplatin and temperature had an apoptotic effect on AGS cells. A. Early apoptosis was enhanced in

cisplatin treated cells. Dashed line represents control rates of apoptosis in untreated cells. Statistical analysis was performed using Mann Whitney test, comparing selected groups to control. * p<0.05. B.

0 .0 0 .5 1 .0 1 .5 2 .0 3 7 Co+ c i s 4 3 Co+ c i s d a ta e q u a li z e d t o c o n tr o l *

Fig.6. Effect of combination therapy on AGS cells (substrates Glu/Ma and Succinate). A-Succinate B-Glutamate/Malate pm ol/ sek/1 m ln cells 0 10 20 30 40 50 60 untreated cisplatin 37 0C 40 0C 43 0C pm ol/sel /1 m ln cel ls 0 20 40 60 80 100 untreated cisplatin 37 0C 40 0C 43 0C RCI 1 2 3 4 5 untreated cisplatin 37 0C 40 0C 43 0C pmol/s ek /m ln 0 10 20 30 40 untreated cisplatin 37 oC 40 oC 43 oC pmol/s el/1 mln ce lls 0 20 40 60 80 100 120 untreated cisplatin 37 oC 40 oC 43 oC RCI 1 2 3 4 5 6 untreated cisplatin 37 oC 40 oC 43 oC

(Glut/Mal)

Mitochondrial´s respiratory function was calculated as described in Methods. We evaluated mitochondrial CI (Glut/Mal)(B) and CII (succinate) (A) dependent respirations (V0), active respiration (VADP), the coupling between respiration and

phosphorylation or RCI (VADP/VCAT), the permeability of the inner (VADP+cyt c) and the

effect of cytochrome c (VADP+cyt c /VADP). Each column represents the mean ± SEM of 4

23

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