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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 7  |  Issue : 4  |  Page : 103-111

Bystander response triggered by doxorubicin-killed dead cells contributes to acquire drug resistance but increasing radiosensitivity In vitro


1 Department of Life Sciences, University of Mumbai; Translational Research Laboratory, Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai, Mumbai, Maharashtra, India
2 Department of Life Sciences, University of Mumbai, Mumbai, Maharashtra, India
3 Department of Life Sciences, University of Mumbai, Mumbai, Maharashtra; Division of Life Sciences, Research Center, Nehru Gram Bharti University, Allahabad, UP; Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India

Date of Web Publication1-Feb-2017

Correspondence Address:
Kaushala Prasad Mishra
Department of Life Sciences, University of Mumbai, Mumbai, Maharashtra; Division of Life Sciences, Research Center, Nehru Gram Bharti University, Allahabad, UP; Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_7_17

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  Abstract 

Introduction: A bystander effect typically refers to the death, altered growth or damage of cells that have not directly received chemotherapy or irradiation. Chemotherapeutic drugs like doxorubicin cause a drastic increase in the number of dead cells more towards the periphery and low towards the centre of the tumor prompting us to test for the existence of a bystander effect in view of the tumor microenvironment.
Materials and Methods: HeLa cervical cancer cells were acutely exposed to doxorubicin to trigger cell death. Bystander HeLa cells in varying amounts were co cultured with fix amount of dead cells. The surviving mutant clones were isolated by serial culturing and checked for morphology, growth pattern and resistance to doxorubicin or radiation.
Results: Co-culture results showed, growth arrest, SA-γ-galactosidase activity, an enlarged cell size, collectively indicating a premature senescent state. Up regulation of p53 and γH2AX indicated a DNA damage response pathway. Co-culturing of a fixed number of dead cells with increasing number of bystander cells showed highest number of clones formed in least number of bystander cells. The individual clones obtained were morphologically altered, reduced proliferation and resistant to doxorubicin. Conversely, clones were sensitive to γ radiation compared to control HeLa cells.
Conclusion: The results suggest that dead cells conferred significant resistance towards drug but not radiation in cloned bystander tumor cells. This point to possible mechanism of drug resistance in vitro, which might explain the success of radiation therapy and cause of frequent tumor recurrence observed in patients undergoing chemotherapy.

Keywords: Bystander effect, dead cells, drug resistance, radiosensitivity


How to cite this article:
Puthli A, Tiwari R, Mishra KP. Bystander response triggered by doxorubicin-killed dead cells contributes to acquire drug resistance but increasing radiosensitivity In vitro. J Radiat Cancer Res 2016;7:103-11

How to cite this URL:
Puthli A, Tiwari R, Mishra KP. Bystander response triggered by doxorubicin-killed dead cells contributes to acquire drug resistance but increasing radiosensitivity In vitro. J Radiat Cancer Res [serial online] 2016 [cited 2018 Nov 19];7:103-11. Available from: http://www.journalrcr.org/text.asp?2016/7/4/103/199310


  Introduction Top


Drug resistance is a major factor that limits the effectiveness of chemotherapy.[1] It ultimately leads to treatment failure in over 90% of patients with metastatic disease.[2] One of the major causes of anticancer drug resistance is the limited ability of drugs to penetrate tumor tissue and to reach all of the tumor cells in a potentially lethal concentration.[2],[3] Drug distribution studies have shown poor penetration for chemotherapy drugs, such as doxorubicin, where primarily high concentrations in the periphery and low concentrations in the center of the tumor were found. Not reaching all of the tumor cells efficiently is a major concern for clinicians.[4] Another concern would be of weak immune system postchemotherapy. During therapy, vast quantities of endogenous dead cells are generated which need to be eliminated from the body, hence clearance of dead cells by an already compromised immune system gets disrupted.[5] Immune competence is crucially required for chemotherapy efficacy relying on complex tumor–host interplay which needs to be overcome to allow the full recovery of immune surveillance and hamper tumor spreading or recurrence.[6] In the tumor, continuous interactions between cancer cells and the surrounding tumor microenvironment actively occur through direct intercellular contact or through secreted signaling molecules.[7] Treatment with drugs such as doxorubicin kills cells by senescence in solid tumors.[8] Senescent cells, while growth arrested, remain metabolically active.[9] They synthesize an array of soluble factors with diverse biological activities.[10],[11],[12] These factors are known to influence the fate of neighboring cells. Previous cell culture experiments have shown that senescent fibroblasts secrete characteristic pro-inflammatory immune cytokines, including interleukin-6 (IL-6) and IL-8, which had the potential to promote bystander cell proliferation and promote cancer.[13] Therefore, signaling components of tissue microenvironments are recognized to profoundly influence cellular phenotypes, including susceptibilities to toxic insults. A previous study has shown how several secreted proteins derived from tumor microenvironment altered the gene expression of WNT16B in the prostate tumor, the mechanism acted in a paracrine manner, while attenuating the effects of cytotoxic chemotherapy in vivo, promoting tumor cell survival and disease progression.[14] Such results highlight a mechanism by which genotoxic therapies given in a cyclical manner can enhance subsequent treatment resistance through cell nonautonomous effects that are contributed by the tumor microenvironment. Then, understanding the cross-talk within the tumor mass microenvironment with a bystander context becomes pivotal in designing strategies for successful treatment and prevention of acquired resistance against anticancer drugs.

A bystander effect has traditionally been defined as the killing or damaging of cells that have not directly received chemotherapy or irradiation, presumably through the diffusion of soluble death-promoting factors from targeted cells.[15] Majority of the experimental protocol involved either medium transfer commonly referred to “conditioned medium” or gap junction-mediated factors.[16] Chemotherapy-associated senescence bystander effect in Michigan Cancer Foundation-7 breast cancer cells was seen with conditioned medium obtained by adriamycin-treated cells.[17] However, until now, all the experimental models have used live cells or conditioned media containing diffusible factors to trigger this phenomenon. Up to our knowledge, there is little or no evidence showing that dead cells might trigger a bystander effect in the absence of any diffusible factor so presumed to be the source of this phenomenon.

In this study, we demonstrate an alternative trigger to the bystander phenomenon. Dead cervical cancer cells provide a mechanism to induce senescence in healthy cervical cancer cells suggesting a novel “dead cell-induced bystander effect.” The experimental basis was to mimic tumor microenvironment of the cancer patients undergoing chemotherapy. HeLa cells were used as a monolayer culture representing the tumor inner cell mass (bystander cells unexposed to drug but surrounded by dead cells after chemotherapy). HeLa cells are a good model system in which to test this as they lack gap junctions responsible for propagation of gap junction-mediated bystander killing present in other cell lines.[18] We discuss our findings in context of senescence-based therapies and mechanism of partial clinical responses to acquired drug resistances that are so frequently observed in cancer patients following chemotherapy.


  Materials and Methods Top


Chemicals and reagents

Thirty millimeters dishes and 96 well tissue culture plates from Becton Dickinson, USA; Dulbecco's modified eagle medium (DMEM), RPMI, penicillin, streptomycin, fetal calf serum (FCS), and trypsin/EDTA were purchased from CellGro (Gibco, Invitrogen, NY, USA). Thiazolyl tetrazolium salt (MTT), doxorubicin from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies used for immunocytochemistry were γH2AX from Calbiochem and (pSer 20) p53 were from BD-Pharmingen. The FITC- or PE-labeled anti-rabbit antibodies were from Sigma-Aldrich.

Cell lines and culture conditions

Cell lines were obtained from the American Type Culture Collection. The HeLa and HT29 cell lines were grown in DMEM containing 10% FCS supplemented with glutamine (2 mmol/L), Hepes (20 mmol/L), streptomycin (10 mg/ml), penicillin (10,000 international units/ml), and sodium bicarbonate (24 mmol/L). The H3255 cell line was grown in a mixture of DMEM and RPMI1640 in equal amounts containing 15% FCS supplemented with glutamine (2 mmol/L), Hepes (20 mmol/L), streptomycin (10 mg/ml), penicillin (10,000 international units/ml), and sodium bicarbonate (24 mmol/L). Cells were grown in 25 ml flasks containing (0.1–1) × 106 cells/ml. The cells were kept in a humidified atmosphere at 37°C containing 5% CO2.

Bystander protocol

For the preparation of dead cells, cells were seeded at densities of 6 × 104 in 30 mm dishes. After 24 h, 5 μg/ml of doxorubicin was added, and dishes kept for 48 h in a CO2 incubator. Dead cells were collected by pouring the supernatant media in a 15 ml centrifuge tube; the remaining dead cells still attached in the dishes were trypsinized and added to the centrifuge tube. The dead cells were washed five times with phosphate-buffered saline (PBS) to remove all traces of doxorubicin by centrifugation at 2500 rpm for 8 min. Dead cells were confirmed by trypan blue dye stain and counted using a hemocytometer.

Plating of bystander cells, cells were seeded at densities of 1.5 × 104, 3 × 104, and 6 × 104 in triplicate 30 mm dishes and kept for 6 h in the CO2 incubator for attaching. To prevent, the cells from doubling 6 × 104 of dead cells were counted and added to each of the recipient dishes after 6 h of seeding; plates were returned to the incubator and kept for 24 h.

After 24 h, dishes were gently washed with PBS twice to remove all the dead cells. Fresh media were added to them and returned back to the incubator. All the dishes were observed daily for morphological changes and pictures captured with the help of Olympus IX70 inverted microscope.

Assessment of growth pattern

HeLa, treated HeLa mother plate, and clones were seeded at a density of 2 × 104 or 6 × 104 in 30 mm dishes (n = 15 for each cell line). Cells were allowed to grow from 24 to 120 h. Daily three dishes were taken on the designated days, washed with PBS, and trypsinized and stained with trypan blue dye to count the number of cells using a hemocytometer. The mean total cell numbers ± standard deviation (SD) presented graphically.

Developing the clones

The mother plate cell line (6 × 104 dead cells treated to 1.5 × 104 bystander cells) was allowed to grow until it was confluent; cells were trypsinized, washed three times with PBS, and suspended at one cell/ml concentration. The cells were then plated (200 μl/well) in 96 well plates. Twenty-four hours after the initial plating, wells containing a single tumor cell were identified and marked. Growth of the single cell-derived colony was monitored over an approximately 2 week period. After the colonies became confluent, they were transferred to 24 well plates and consequently to T-75 flasks and 100 mm plates. Clones were named based on their position where a single cell was found in the 96 well plates.

Identification of senescent cells and markers of DNA damage

The SA-β-galactosidase activity assay was employed for the identification of senescent cells on 6 × 104 bystander treated cells at a period of 96 h as described previously.[19] The markers of DNA damage were performed as described previously.[20] Briefly, HeLa cells were seeded on coverslips at a density of 6 × 104 cells and allowed to grow overnight (16 h) and subjected to treatment with 6 × 104 dead cells for 12 h in duplicate experiments. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized in 0.2% Triton for 30 min, blocked in 3% bovine serum albumin for 1 h, and immunostained overnight with p53 and γH2 AX-specific antibodies against DNA damage response (DDR) proteins. Cells were immediately mounted on slides with Vectashield, and images were acquired through Spectral Bio-Imaging System (Applied Spectral Imaging, Israel).

Evaluation of sensitivity to doxorubicin and γ-radiation

For testing the drug sensitivity, HeLa and clones were seeded at density of 5,000 cells/well in a 96 well tissue culture plates. Cells were treated with four different concentrations of doxorubicin (0.001/µg) for 48 h in a CO2 incubator. After the desired time of 48 h, the wells were washed with PBS once, and MTT (0.1 mg/well, Sigma, USA) was added to all the wells followed by incubation for 4 h at 37°C. The formazin crystals formed were solubilized by incubating the cells with 200 µl dimethyl sulfoxide. The absorbance of the solution was measured at 570 nm, using a microplate reader (BioTek Instruments, USA). % survival was calculated using the formula



Similarly, for radiosensitivity, exponentially growing HeLa and clones were seeded overnight at density of 5,000 and 10,000 cells/well in a 96 well tissue culture plates. After 12 h, 96 well plates were irradiated at 0.5, 2, and 10 Gy using a cobalt-60 teletherapy source (Theratron-780 machine installed at Advanced Centre for Treatment, Research and Education in Cancer, India, at a dose rate: 1.95 Gy/min.). Thermoluminescent dosimeters were used to confirm that the appropriate dose was delivered. The plates were returned to the CO2 incubator immediately after irradiation and processed after 48 h using the MTT protocol mentioned above.

Statistical analysis

All results are expressed as mean ± SD of three experiments with triplicates for each sample. The Student's t-test was done to determine the statistical significance of the difference in the absolute values of the number of cells for growth rate and % survival between the treated and nontreated groups. The value of P < 0.05 was considered significant.


  Results Top


Morphological alterations of bystander cells with incubation time

The morphological changes in HeLa bystander cells seeded at different cell densities of 1.5 × 104, 3 × 104, and 6 × 104 treated with 6 × 104 dead cells were seen in [Figure 1], where pictures of bystander cells at early and late time points of 48 h and 6 days were captured. Forty-eight hours have shown early signs of senescence, such as cell flattening and cell cycle arrest when compared with the controls in all the ratios. Same samples kept for 6 days show adverse morphological changes resembling premature senescence, where extreme cell flattening is seen in 6 × 104, little less flattening with few transformational changes seen in 3 × 104, and least cell flattening with maximum transformational changes seen in 1.5 × 104 bystander cells, hence named mother plate. [Figure 2]a confirmed the accumulation of senescence-specific β-galactosidase [Figure 2]b persistence of γH2AX foci and [Figure 2]c nuclear p53 at 96 h in 6 × 104 HeLa bystander cells confirming a DNA damage response pathway. Similar morphological changes were observed in [Figure 3] with H3255 cell line. However, HT29 cells showed signs of cell cycle arrest but not the characteristic premature senescence morphology.
Figure 1: The panel shows cell morphological changes induced by dead cells to bystander HeLa cells seeded at different densities (1.5, 3, 6) × 104. Where 6 × 14 dead cells were cocultured for 24 h. The dead cells were removed by washing dishes with phosphate-buffered saline twice and fresh media added; they were photographed using inverted microscope (×10) at 48 h and 6 days after treatment. Horizontal rows show the number of bystander cells seeded and vertical rows depict control and treated cells, arrows indicate premature senescent cell

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Figure 2: Accumulation of (a) senescence-specific μ-galactosidase (blue stain) (b) persistence of γH2AX foci and (c) nuclear p53 at 96 h in 6 × 104 HeLa bystander cells seen under × 20

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Figure 3: Morphological changes induced by dead cells in two different bystander cell lines, human colon adenocarcinoma (HT29), and human lung adenocarcinoma (H3255). Images were captured after 7 days of treatment

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Viability of bystander cells treated with dead cells

The loss of viability decreased in bystander cells treated with dead cells estimated after 6 days seen in [Figure 4], where 60,000 and 30,000 seeded cells showed a highly significant decrease in viability to 5 × 104 (P < 0.0001) and 7 × 104 (P < 0.0001), respectively, meaning the cells were unable to proliferate. Similarly, 15,000 bystander cells showed a staggering 45% loss in viability (P < 0.0001).
Figure 4: Viability counts in bystander HeLa cells after 6 days. Cells were stained with trypan blue dye and counted using a hemocytometer. Only 15,000 bystander controls are shown, due to over confluency the other two controls were not considered

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Growth rate of HeLa cells and mother plate

We observed that mother plate cells were growing at a slower rate as compared to the HeLa cells seen in [Figure 5]. Comparing the growth at different time intervals between HeLa and mother plate, we observed that mother plate showed a significant difference in growth even at an early time point of 24 h (P > 0.01) proliferating more slowly as time progressed to 96 h (P > 0.0001).
Figure 5: Comparative growth curve measurement of control HeLa cells with mother plate

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Morphological changes and growth rate in clones

Morphological differences observed between HeLa cells, mother plate, and clones were seen in [Figure 6]. Mother plate cells were more refractive, striated, and grow more compacted than the HeLa cells. The H-1 clone looks more striated and refractile, E-5 clone looks more refractive and rounded in shape, whereas 2.E-5 was nonrefractive and round. G-3 looked slight similar to HeLa cells.
Figure 6: Cell morphology observed in control HeLa, mother plate, and clones (2E-5, E-5, H-1, and G-3)

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The comparative growth rate between HeLa cells and clones was seen in [Figure 7]; we could observe that all the clones grew at different rates among themselves; comparatively, clone G-3 and H-1 grew slowest.
Figure 7: Comparative growth curve measured for a period of 120 h between HeLa with clones (2E-5, E-5, H-1, and G-3)

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Clone sensitivity to doxorubicin and gamma-radiation

[Figure 8] shows the percentage survival as a function of doxorubicin concentration (0.001–1 μg), where HeLa cells have shown drug sensitivity in a dose-dependent manner, showing a significant decrease of 17% (P < 0.05) at lowest concentration of 0.001 μg compared to untreated cells; however, all the clones showed resistance at same concentration (P > 0.05). As concentration increased to 0.01 μg, we see further decrease in HeLa cells (P < 0.05), whereas all the clones decreased insignificantly (ns) compared with untreated controls. Further increase in concentration till 0.1 μg resulted in some of the clones being sensitive equally to HeLa cells except H-1 and G-3 (P < 0.01). Drug resistance was not observed beyond 1 μg.
Figure 8: Chemosensitivity study between HeLa cells and clones treated with increasing concentrations of doxorubicin (0.001–1 μg) for 48 h

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Results from [Figure 9] depict the radiosensitivity between HeLa and Clone cells given various doses of γ-radiation. The doses ranged from a low dose 0.5 Gy, therapeutic dose of 2 Gy, and high dose of 10 Gy. Results showed that both HeLa cells and the clones showed no significant sensitivity toward the low dose of 0.5 Gy. Significant decrease in cell survival was seen in HeLa cells given 2 Gy dose (P < 0.05). Similar sensitivity was seen in clone 1E-5 by almost 10% (P < 0.05). Maximum sensitization was seen in clones 2E-5 and G-3 showing a reduction of 21% (P < 0.01) and 32% (P < 0.01), respectively, with 2 Gy dose. However, clone H-1 showed no sensitivity at 2 Gy dose. The cell survival of H-1 clone decreased by 16% (P < 0.01) when given a high dose of 10 Gy. At high dose, an overall increase in sensitization was seen in all the cells.
Figure 9: Radiosensitivity study between HeLa cells and clones exposed to increasing doses of γ-radiation (0.5–10 Gy) for 48 h

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  Discussion Top


In the first series of experiments, we have attempted to address the question whether dead HeLa cells were competent to effect healthy recipient cells (i.e., bystander killing). The coculture results contained in [Figure 1] clearly showed early and late time point observations (48 h and 6 days), where cells that were killed by doxorubicin were capable of eliciting a bystander response to healthy recipient cells. Our morphological observations suggest the signs of cell cycle arrest and premature senescence beginning 48 h gradually increasing in intensity till 6 days. These results point out that communication signals were received by healthy recipient cells, resulting in cytotoxic effects. We further observed that cells became large, flat, multinucleated, refractile, and spindle-shaped. A flat-cell phenotype is commonly seen in cells undergoing H-RAS V12-induced senescence.[21] Our results showed extensive vacuolization similar to those reported by others.[21] Similarly, cells senescing due to BRAF E600 expression or the silencing of p400 acquired a more spindle-shaped morphology [22],[23] seen in our results. It appears that multiple pathways might be at hand in generating morphological diversity. In another coculture study, researchers showed that apoptotic HeLa cells treated with three different apoptogens (TNF, staurosporine, and H2O2) generated apoptotic signals that were transmitted to intact cells through the medium without direct contact.[24] This means that early or late apoptotic cells show specificity toward an apoptotic pathway in bystander cells. The major differences observed were a senescence signaling mechanism induced by dead cells in our experiments. However, dead cells were in direct contact for 24 h and later washed. Interestingly, when dead cells were continuously exposed for 72 h with cells, it showed an enhanced deleterious effect as compared to 24 h exposure (data not shown), such similar results were seen by researchers who showed that the frequency of bystander MRC5 and BJ cells positivity for senescence markers such as β-galactosidase (Sen-β-Gal) activity was increased after coculture with senescent-induced cells for 15 or more days.[25] Our result seen in [Figure 2]a supports this and most probable that the source of senescent signal lays in the dead cell. We also noticed that cell number of the bystander cells played a crucial role in the extent of cellular damage taking place; given the fact that the strength of the signal being the same based on the same number of dead cells added to all bystander cells, we saw an equal number of bystander cells to dead cells showed the maximum amount of damage as compared to quarter the amount of bystander cells showing the least damage. This clearly indicates that the bystander cells are taking part and augmenting the senescence signaling process, whereby contributing to the overall strength of the signal. Similar results have shown that senescent cells induced a DDR, characteristic for senescence, in neighboring cells through gap junction-mediated cell–cell contact and processes involving reactive oxygen species.[25] However, HeLa cell line lacks gap junctions, and DDR was possibly mediated through a secretary phenotype, nevertheless a continuous presence of γH2AX foci and phosphorylated (phospho-Ser20) p53 were seen in [Figure 2]b and [Figure 2]c, respectively, indicated prolonged persistence of DDR in bystander cells. We have observed that decreasing the number of dead donor cells resulted in an increase in cell survival of bystander cells (data not shown). Therefore, our experiments have shown that the intensity of cellular damage to bystander cells depends on the cell number of both, dead donor and bystander recipient cells in vitro.

Apart from the HeLa cell line that lacks gap junctions, we confirmed that this effect was exhibited in several cell lines, such as mouse fibroblast (NIH3T3), mouse melanoma (B16-F10), human fibroblast (MRC-5), human colon adenocarcinoma (HT29), lung adenocarcinoma (H3255), and found similar effects, confirming that it was not restricted to HeLa cells. In this paper, we have shown the results of chemoresistant HT29 and H3255 as seen in [Figure 3]. We found that all of the cell lines showed cell cycle arrest and premature senescence except in the HT29 cell line that showed cell cycle arrest but did not show typical premature senescence morphology. This could be explained such that HT-29 has a mutated p53 gene that results in nonfunctional protein [26] mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence,[9],[10] nevertheless we were able to show that bystander HT29 cells reacted differently to its dead cells in an unknown mechanism which needs further investigation.

Since senescent cells were observed in treated bystander HeLa cells, we have investigated the viability of treated cells after 6 days to check if they could divide and proliferate. Trypan blue dye stained cell count in [Figure 4] has shown that 6 × 104 bystander cells were unable to divide after 6 days indicating that all of the cells were senescent. Similarly, in the 3 × 104 bystander cells divided only 2-fold. Interestingly, maximum number of cells were able to proliferate in the 1.5 × 104 bystander cells, but not as efficiently as the control untreated cells, this could be interpreted such that although the dead cell signal strength being the same, lesser the number of bystander cells could escape the senescence-induced senescence cycle (nonautonomous effects) and proliferate.

The treated bystander dish (1.5 × 104) was able to divide and proliferate, and we have named it mother plate and cultured it for an additional 25 passages, to check if cells would reenter senescence or repair itself to a normal growth rate. Results from growth rate experiment in [Figure 5] show that mother plate grows at a slower rate as compared to the control HeLa cells, suggesting that dead cells could slow down the rate of proliferation in neighboring tumor cells.

On closer inspection, it was noticed that there were considerable morphological differences seen between control HeLa and the mother plate. It has been previously shown that senescent factors alter the genotype and phenotype in cells. Human tumor cells very likely express a senescence-associated secretory phenotype (SASP) after chemotherapy. They speculated that components of chemotherapy-induced SASPs, particularly the high levels of inflammatory cytokines, might contribute to the debilitating effects of DNA-damaging chemotherapy. They hypothesized that SASPs might also fuel development of secondary cancers by creating a local tissue environment that is permissive for the growth and progression of cells that acquire therapy-induced mutations, and cells fail to sense or die.[26] Our findings support the idea that senescent cells can create a tissue microenvironment that promotes multiple stages of tumor evolution.

To test if senescent cells contribute toward tumor evolution, we derived 16 individual clones from the mother plate out of which four clones were randomly selected and screened for three parameters: Morphology, growth rate, and sensitivity toward drug or radiation. Our observations as seen in [Figure 6] clearly indicate that all the clones show a pronounced morphological diversity from each other, H-1 clone looks more striated and refractile, E-5 clone looks more refractive and rounded in shape, whereas 2.E-5 was nonrefractive and round. G-3 looked more or less similar to HeLa cells.

Since tumor clones differed in morphology, we hypothesized that stress-induced mutational changes might affect growth rate in individual clones; therefore, we calculated growth rate for a period of 5 days. The results given in [Figure 7] have shown that growth was different between all the clones; however, interestingly, it was less than that of HeLa cells, meaning dead cell-induced senescent factors slow down the proliferation of stress-induced bystander cancer clones. Controversy regarding tumor suppression or promotion by SASP's still exists which needed to be addressed. Some argue that cellular senescence restrains cancer by imposing a cell-autonomous block to the proliferation of oncogenically damaged/stressed cells. In addition, each SASP factor may have effects that depend on the cell and tissue context.[27] For example, several researchers have shown that the IL-6, IL-8, and plasminogen activator inhibitor-1 that is secreted by senescent fibroblasts can promote tumor suppression by reinforcing the senescence growth arrest induced by activating oncogenes or oxidative stress.[28],[29],[30] However, IL-6 and IL-8 have also been shown to promote malignant tumorigenesis in cooperation with certain activated oncogenes.[31],[32] Our results support both the claims and have shown a link between tumor suppression through senescent growth arrest and resistant tumor progression through a bystander model.

Chemoresistance is a major stumbling block to the successful treatment of cancer patients, since tumor cells, either fail to reduce in size after toxic insult or cancer reoccurs subsequent to an initial “positive” response.[33] We hypothesized that dead cell-induced senescent factors could generate mutant clones within the tumor microenvironment, making them resistant to successive drug treatment. To ascertain the origin of cancer recurrence arising from the same tumor, we decided to check drug sensitivity between HeLa cells and clones derived from mother plate mimicking the patient undergoing successive chemotherapy cycle. We treated the cells with varying concentrations of doxorubicin (0.001–1 µg) for 48 h. Results from [Figure 8] clearly showed that HeLa cells were sensitive to doxorubicin in a concentration-dependent manner; on the contrary, the clones were more resistant. The limit of quantitation of doxorubicin in human blood plasma is 1.0 ng/ml.[34] Therefore, resistance by the clones was observed within the clinical physiological range.

To test if chemoresistance occurred exclusively through the bystander phenomenon, we derived 18 clones from untreated HeLa cells (control) and found no resistance to doxorubicin (data not shown).

A correlation was seen between slow growth rate and drug sensitivity, where slower growing clones displayed maximum resistance to doxorubicin. Slower-replicating cancer cells are less likely to be destroyed by chemotherapy and yet which are responsible for reseeding and fueling the growth of the tumor itself with a theoretical limitless resupply of daughter cells.[35]

HeLa cells are known to be radioresistant.[36] To understand if dead cells were able to modify the radiation response in bystander cells, we exposed the clones to γ-radiation. Our results in [Figure 9] showed that therapeutic dose of 2 Gy was successful in sensitizing most of the tumor clones except H-1 clone whose sensitivity could be increased with higher dose of radiation.


  Conclusion Top


Our results have shown that there is a vast possibility that within the tumor microenvironment doxorubicin-killed cells could generate stress-induced bystander progeny cells making them resistant to successive doxorubicin treatment. Our results also suggest that radiotherapy would be more efficient in treating patients with acquired drug resistance. Better understanding of the resistance mechanisms is highly warranted to improve the clinical efficiency of current regimens leading toward new therapeutic targets and development of better anticancer strategies.

Acknowledgments

Authors like to thank Mr. Naveen Khare and Ms. Sonal Channale for their technical help. We further extend our thanks to Dr. Pradyumna Mishra, Scientific officer and Dr. Indraneel Mittra, Head, Translational Research Laboratory, for their valuable suggestions and encouragement during this work.

Financial support and sponsorship

Nil.

Conflicts oF interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]


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