|Year : 2015 | Volume
| Issue : 4 | Page : 904-910
Chelerythrine delayed tumor growth and increased survival duration of Dalton's lymphoma bearing BALB/c H 2d mice by activation of NK cells in vivo
Sanjay Kumar, Munendra Singh Tomar, Arbind Acharya
Department of Zoology, Faculty of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Web Publication||15-Feb-2016|
Department of Zoology, Faculty of Science, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Aim: The aims of the present investigation were to evaluate the antitumor effect of chelerythrine (CHE) on in vivo growth and survival duration of BALB/c (H 2d ) mice bearing Dalton's lymphoma (DL) and enhanced function of tumor associated NK cells (TANK cells).
Materials and Methods: BALB/c (H 2d ) mice at 8-10 weeks of age of either sex were used. Increasing concentration of CHE (1.25, 2.5, and 5.0 mg/kg), staurosporine (0.625, 1.0, 1.5, and 2.0 mg/kg) and cyclophosphamide (25, 50, 100, and 200 mg/kg) were administered intraperitoneally and tumor regression and survival duration of tumor bearing host were determined, and thereafter expression of NKG2D and NKG2A on TANK cells were detected.
Results: Our results show that treatment with 2.5 mg/kg of CHE results in a significant reduction in mean tumor volume and increased survival duration of DL bearing BALB/c (H 2d ) mice when compared to control. Activating receptor NKG2D on TANK cells were observed upregulated in contrast to inhibitory receptor NKG2A.
Conclusions: CHE reduced mean tumor volume and increased survival duration of DL bearing BALB/c (H 2d ) mice. Increased expression of activating receptor NKG2D on TANK cells results in recovery of immunosuppression during tumor progression. Therefore, CHE could be a potential anticancer therapeutic agent that may be used to replace chemo-radio-therapy in future.
Keywords: Chelerythrine, Dalton′s lymphoma cells, survival duration, tumor associated NK cells, tumor volume
|How to cite this article:|
Kumar S, Tomar MS, Acharya A. Chelerythrine delayed tumor growth and increased survival duration of Dalton's lymphoma bearing BALB/c H 2d mice by activation of NK cells in vivo. J Can Res Ther 2015;11:904-10
|How to cite this URL:|
Kumar S, Tomar MS, Acharya A. Chelerythrine delayed tumor growth and increased survival duration of Dalton's lymphoma bearing BALB/c H 2d mice by activation of NK cells in vivo. J Can Res Ther [serial online] 2015 [cited 2021 Apr 12];11:904-10. Available from: https://www.cancerjournal.net/text.asp?2015/11/4/904/143342
| > Introduction|| |
Chelerythrine (CHE) is a selective inhibitor of protein kinase C (PKC), which shows a wide range of biological activities include antitumor, antiinflammatory and antiproliferative against many cancers; therefore, it can be used as an effective anticancer agent. , Anticancer activity of CHE arises from its ability to interact with proteins and DNA via addition of nucleophilic groups (e.g. thiol groups) to the iminium bond, by ionic interaction or by DNA intercalation.  Quaternary nitrogen atom with fully hydrogenated rings is the most peculiar characteristic of benzophenenthridine alkaloids includes CHE.  In addition, CHE is selectively cell permeable inhibitor of serine/threonine (ser/thr) kinases which competes for the conserved catalytic domains of PKC. PKC, a member of ser/thr kinase family consist 12 isozymes such as conventional PKCs, novel PKCs and atypical PKCs that have shown distinct domain organization.  Conventional PKCs play a major role in cell growth, proliferation, differentiation, and carcinogenesis under stress.  Structurally they have an N-terminal regulatory domain, which activate by tumor promoter phorbol ester binding domains and a C-terminal catalytic domain, which binds to both the adenosine triphosphate and substrate have autophosphorylation sites. ,, The C-terminal domain considered to be the main target for PKC inhibition. Unlike staurosporine, 7-hydroxyl-staurosporine, and UCN-01 CHE does not inhibit other kinases or phosphatizes. ,,
Non-specific inhibition of PKC functions by a microbial alkaloid, staurosporine leads to characteristic hallmark of apoptosis in many cancer cells. , Existing literature suggest that inhibition of PKC results in activation of apoptotic cascades in nucleated mammalian cells through distinct phase of their development.  Therefore, pharmacological inhibition of PKC might be competent to alter biochemical events associated with apoptosis. Little about the selectivity of PKC inhibitors is known, and potential clinical applications for selective inhibition of PKC need to be investigated in details. Apoptosis results in altered biochemical events such as release of free Ca ++ from intracellular stores, release of cytochrome-c, withdrawal of pro-survival factors (Bcl2 and Bcl-xL) that induce the activation pro-apoptotic factors (Bax and Bcl-Xs). ,,,, Moreover, CHE has been identified as a potent inhibitor of Bcl-xL function in human SH-SY5Y and MCF-7 cell lines.  In the present report, we show that CHE delayed tumor growth and progression and increased survival duration of Dalton's lymphoma (DL) bearing BALB/c H 2d mice. However, previous reports failed to demonstrate in vivo anti-cancer effect of CHE in leukemia model. , We found that in vivo administration of CHE (2.5 mg/kg) showed significant inhibition of tumor growth and progression in BALB-c/H 2d mice which results in increased survival duration of tumor bearing host as compared to staurosporine. In addition, expression of NKG2D on tumor associated NK (TANK cells) was found upregulated in contrast to NKG2A. Our previous reports suggest that apoptosis was the predominant mechanism of CHE -induced DL cell killing in vitro.
| > Materials and methods|| |
BALB/c (H 2d ) mice of either sex were used at 8-10 weeks of age. Mice were kept in utmost care in an approved animal room facility under the ethical guidelines for the use of animals in scientific research by Indian Council of Medical Research, New Delhi.
For tumor modal, spontaneous occurring T-cell lymphoma designated as DL were selected owing to the property of its highly invasive and deleterious effect on the host's immune system. Groups of mice were injected (ip) with 1.0 × 10 6 DL cells/mL in sterile Phosphate Buffer Saline (PBS) and allow to grow.
Chelerythrine, staurosporine and cyclophosphamide monohydrate were purchased from Sigma Chemical Company, Bengaluru, India. RPMI-1640 culture medium was purchased from HiMedia, Mumbai, India and Fetal Bovine Serum was obtained from Gibco, India. PBS-10x was purchase from Ambion, India. NK cells receptor antibodies NKG2D-FITC, NKp46-FITC, Ly49A-FITC, and Ly49D-FITC were purchased from Biolegend, San Diego, CA, USA, and Ly49C-FITC and NKG2A-FITC were obtained from eBioscience, San Diego, CA, USA. Mouse CD49b positive selection kit was purchased from Stem Cell Technology, Vancouver, Canada. All other chemical stated otherwise were obtained either from Sigma Chemical Company, Bengaluru, India or Super Religare Laboratories, Mumbai India.
Single cell suspension of DL cells (1.0 × 10 6 DL cells/mL) was prepared and injected intra-peritoneally into the right hind limbs of BALB/c (H 2d ) mice and allow to grown for 10 days, at this time animals were sorted into treatment and control groups. The animals were serially monitored by manual palpation for evidence of tumor growth. Initially mean tumor volume was observed (~3.0 × 10 7 cells/mL) after 10 days of post DL cells transplantation. Experimental Group 1 (n = 10) were administrated with different concentration of staurosporine and CHE along with cyclophosphamide (0.625 mg/kg, 1.5 mg/kg, and 25 mg/kg) freshly prepared in DMSO and Group 2 administered with (1.0 mg/kg, 2.5 mg/kg and 50 mg/kg) concentration of aforementioned chemical. Further, Group 3 was treated with (1.5 mg/kg of staurosporine, 5 mg/kg of CHE and 100 mg/kg of cyclophosphamide and Group 4 was treated with (2.0, 5, and 200 mg/kg) concentrations of staurosporine, CHE and cyclophosphamide, respectively. Cyclophosphamide was used as a positive control. The doses were administered according to host body weight, five dose at alternate days scheduled from day 11 (D 11 ) to day 21 (D 21 )(D 11 to D 21 ), two doses after 3 days intervals from day 22 (D 22 ) to day 27 (D 27 ) and one dose after 7 days intervals (if necessary) at day 34 (D 34 ) were administered. Control group of mice (n = 10) were administrated with sterile PBS (1x) in similar experimental conditions.
Three treatment modalities were adopted to evaluate the anticancer effect of CHE and staurosporine on in vivo growth of DL cells in BALB/c (H 2d ) when compared to control. Tumor was induced by injection of 1.0 × 10 6 cells/mL intra-peritoneally into right hind limbs of BALB/c (H 2d ) mice. When tumor becomes established, mice were shorted into four treatment groups along with respective control. Treatment was started from day 11 (D 11 ) and Group 1 was administered with CHE (1.25 mg/kg), staurosporine (0.625 mg/kg) and cyclophosphamide (25 mg/kg), and administration with staurosporine (1.0 mg/kg), CHE (2.5 mg/kg) and cyclophosphamide (50 mg/kg) were followed in Group 2. Group 3 was treated with CHE (5 mg/kg), staurosporine (1.5 mg/kg) and cyclophosphamide (100 mg/kg) and 2.0 mg/kg of staurosporine, 5.0 mg/kg of CHE and 200 mg/kg of cyclophosphamide were administered in Group 4. Treatment with CHE, staurosporine and cyclophosphamide were continued and mean tumor volume in terms of DL cell count per mL was determined.
Kaplan-Meier survival analysis was followed to compare the time to develop tumor in treatment as well as control groups. Aforesaid method was applied to know whether CHE and staurosporine are able to regret established tumor in BALB/c (H 2d ) mice. First, an overall test was performed thereafter pair wise comparison were carried out. Treatment schedule was split to minimize the systemic toxicity and Group 1 was administered with staurosporine (0.625 mg/kg), CHE (1.25 mg/kg) and cyclophosphamide (25 mg/kg), Group 2 was administered in the presence of 1.0 mg/kg of staurosporine, 2.5 mg/kg of CHE, 50 mg/kg of cyclophosphamide, Group 3 was administered with staurosporine (1.5 mg/kg), CHE (5 mg/kg) and cyclophosphamide (100 mg/kg) and Group 4 were administered with 2.0 mg/kg of staurosporine, 5 mg/kg of CHE and 200 mg/kg of cyclophosphamide. Treatment schedule was similar as described previously and all treated and control groups were monitored sincerely and the numbers of survival host were recorded. The median survival duration of DL bearing BALB/c (H 2d ) mice was determined and expressed in terms of percent survival.
Tumor associated NK cells were isolated using Magnetic Associated Cell Sorter (MACS-EasySep Magnet "Stem Cell, Chennai, India") according to the manufacturer's instruction.  Spleenocytes (1.0 × 10 8 cells/mL) were incubated with mouse CD49b PE labeled antibody for 15 min at 15-25°C and EasySep PE-selection cocktail was added and incubate for 15 min at 15-25°C. Further, cells were incubated with EasySep magnetic nanoparticles mixed gently, and the tube was placed into the magnet for 5 min. TANK cells (>98% pure) were stained with anti-NKG2D-FITC and anti-NKG2A-FITC conjugated antibodies and expression of NKG2D and NKG2A were analyzed by Flow cytometry (FACS).
Data were analyzed by one-way ANOVA followed by Bonferroni method as posttest. Data were expressed as mean ± standard error. The data were considered to be significant at P ≤ 0.05 as applicable. All statistical analyses were done on Sigma plot Version 12.0 (Systat Software Inc., San Jose, CA, USA).
| > Results|| |
Selective studies are existing which demonstrate that CHE exhibited in vivo anticancer and anti-proliferative effect against several cancers of human origin such as WEHI-231 and SQ-20B.  This study was conducted to ensure whether PKC inhibitors, CHE and staurosporine delayed in vivo growth and progression of DL cells in BALB/c (H 2d ) mice. On the basis of existing literature, single dose may induce systemic toxicity therefore dosing schedule was split to minimize systemic toxicity. Dosing schedule was followed for DL bearing BALB/c (H 2d ) mice as described in materials and methods.  Early studies suggest that a single dose of CHE did not result in antileukamic effect in p388 mouse leukemia model, however significant anticancer or antiproliferative effects were observed on SQ-20B and HNSCC cells. ,,, Antiproliferative activity of CHE on this nonregressing tumor model could be caused either by suppression of proliferation or by induction of apoptosis. To know this cause, the rate of reduction in mean tumor volume was determined in treated as well as control groups of mice. Results show that Group 1 was unable to produce significant growth inhibitory effect after treatment with staurosporine (0.625 mg/kg) and CHE (1.25 mg/kg) as compared to control [Figure 1]a. Cyclophosphamide (25 mg/kg) was used as a positive control, an established anticancer drug and being used in clinical practice [Figure 1]a. Group 2 was treated with 1.0 mg/kg of staurosporine, 2.5 mg/kg of CHE and 50 mg/kg of cyclophosphamide showed a remarkable difference in the mean tumor volume of treated group as compared to control [Figure 1]b. This difference was observed highly significant after administration with CHE (2.5 mg/kg) as compared to staurosporine and control [Figure 1]b; however, staurosporine (1 mg/kg) failed to produce significant growth inhibitory effect against progressive growth of DL cells [Figure 1]b. Further, Group 3 was treated in the presence of staurosporine (1.5 mg/kg), CHE (5.0 mg/kg) and cyclophosphamide (100 mg/kg), but no remarkable difference in the mean tumor volume was observed between treatment and control groups [Figure 1]c. Administrated with 2.0 mg/kg of staurosporine, 5 mg/kg of CHE and 200 mg/kg of cyclophosphamide in Group 4 show that high dose of CHE, staurosporine and cyclophosphamide did not results in a significant reduction in mean tumor volume [Figure 1]d. Initial tumor volume was observed almost similar in all treatment groups, but the difference was visible after 15 days of post treatment as described previously. Among four, Group 2 showed gradual, but highly significant rate of tumor regression when compared to other groups [Figure 1]. In addition, Group 1 showed early death of tumor bearing mice that may be due to tumor burden [Figure 1]. In addition, Group 3 and Group 4 were also died earlier than other groups even after low tumor volume suggest that death may occur due to high dose of CHE, staurosporine and cyclophosphamide, which may cause systemic toxicity/injuries.  These findings suggest that CHE treatments results in significant inhibition of tumor growth in BALB/c (H 2d ) mice.
|Figure 1: Effect of chelerythrine (CHE) on in vivo growth of Dalton's lymphoma (DL) cells in BALB/c (H2d) mice. Mice were injected with (1.5 × 106 DL cells/mL) intraperitoneally. After 10 days of post DL cells transplantation mice were shorted into experimental and control groups and each respective group of mice were administered with increasing concentration of CHE, staurosporine and thereafter tumor volume was determined. Cyclophosphamide was used as positive control, which is an established drug. Tumor volume of each group was determined in terms of DL cells count per mL. Data were expressed as mean ± standard error of three individual experiments. The data were taken as significant at P ≤ 0.05 as applicable|
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To determine whether PKC inhibitors increased survival duration of DL bearing mice, Kaplan-Meier survival analysis was followed to compare the survival duration of treated groups with their respective control. Although, growth rate of DL cells is much higher (tumor volume doubling time ~5 days), but it has some limitations. This faster growth rate of DL cells is mainly driven by a high rate of DL cell proliferations (~30% growth fractions for DL cells in vivo). Mice were treated with staurosporine (0.625 mg/kg) showed low duration of survival as compared to control [Figure 2]a. However, CHE (1.25 mg/kg) showed increased survival duration, but it was not significant when compared to control [Figure 1]a. Mice treated with different concentration of cyclophosphamide at (25, 50, 100, 200 mg/kg) were used as a positive control [Figure 2]a-d. In contrast to Group 1, Group 2 in the presence of staurosporine (1.0 mg/kg), CHE (2.5 mg/kg) and 50 mg/kg of cyclophosphamide showed significantly increased survival duration of DL bearing BALB/c (H 2d ) mice when compared to control [Figure 2]b. The survival duration of CHE treated Group 2 can be considered almost similar to that of positive control (cyclophosphamide). However, Group 3 failed to achieve a significant level of host survival [Figure 2]c. Therefore, Group 3 and Group 4 showed poor rate of survival duration even after high dose of CHE and staurosporine as compared to Group 2. Present results correlated with previous findings that CHE delayed DL growth in those mice that had developed tumors, a rapid and complete tumor regression occurred from the time of tumor onset at days 18, 21 until days 34 after tumor cell implantation.  One out of 10 (10%) after 55 days in CHE treated group of BALB/c (H 2d ) mice and two out of 10 (20%) after 65 days in cyclophosphamide treated group of mice were shown increase rate of mortality [Figure 2]b. CHE treatment either at a lower concentration or at a higher concentration did not results in a significant anti-cancer effect and/or increase mortality rate with 10 out of 10 (100%) animals developing more asitic fluid by day 22-32 for both doses. Similarly, cyclophosphamide treatment with a low dose and with a high dose did not results in a significant increase of survival duration of tumor bearing host 10 out of 10 (100%) animals developed asitic fluid by days 34 [Figure 2]c and d. Our results corroborate with previous findings that CHE (2.5 mg/kg) and cyclophosphamide (50 mg/kg) increased survival duration of tumor bearing host, which was observed statistically significant (P < 0.001). Efficacy validation of CHE in HNSCC cell line and cyclophosphamide in T50/80 tumor cells were studied previously and showed similar effects.  These findings suggest that CHE (2.5 mg/kg) increased survival duration of tumor bearing host up to 55 days while cyclophosphamide (50 mg/kg) increase host mortality rate up to 65 days which is significantly higher.  In contrast, treatment with staurosporine failed to regret tumor in vivo at any given concentration, while in vitro evidence suggests that 1 μM of staurosporine induced apoptosis in many cancers and their cell lines. 
|Figure 2: Effect of chelerythrine (CHE) on survival duration of Dalton's lymphoma (DL) bearing BALB/c (H2d) mice. Mice were administered with increasing concentration of CHE and staurosporine after established tumor, survival duration of DL bearing BALB/c (H2d) mice was determined with the help of Kaplan-Meier survival plot. We used cyclophosphamide as a positive control. Mice without any treatments were used as negative control. Survival duration of each group was expressed in terms of median survival. Three independent experiments were represented as mean ± standard error and taken significant at P ≤ 0.05 as applicable|
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Although, existing reports suggest that TANK cells function impaired during tumor progression include decrease cytotoxic activity, downregulation of activating receptor expression and intracellular signaling molecules, defective proliferation, poor infiltration, decreased cell counts, and decreased cytokine production. [20-37] In the present study, characterized and purified NK cells were counter stained with rat anti-mouse NKG2D-FITC and NKG2A-FITC and observed under flow cytometry. We found that the administration of CHE (2.5 mg/kg) and cyclophosphamide (50 mg/kg) results in increased expression of activating receptor NKG2D on TANK cells surface as compared to control; however, treatment with staurosporine (1.0 mg/kg) was not [Figure 3].  In contrast to activating receptor NKG2D, inhibitory receptor NKG2A expression was found downregulated after administration of CHE (2.5 mg/kg) and cyclophosphamide (50 mg/kg) as compared to control [Figure 4]. Basal level expression of inhibitory receptor NKG2A was observed after treatment with staurosporine (1.0 mg/kg) [Figure 4]. Results show that the function of TANK cells were seems to be restored after administration of CHE (2.5 mg/kg) even late onset of tumor which may be important for the recovery of immunosupression caused by tumor progression but further investigation is needed to explored the exact mechanism underlie.
|Figure 3: Effect of chelerythrine (CHE) on the expression of NKG2D on tumor associated NK (TANK) cells. Mice were administered with CHE (2.5 mg/kg), staurosporine (1.0 mg/kg) after 24 (control)/34 (experimental) days of post Dalton's lymphoma (DL) cells transplantation, TANK cells were isolated using Magnetic Associated Cell Sorter (MACS-EasySep Magnet) and subjected to stained with specific anti-NKG2D-FITC antibody. Expression of NKG2D on TANK cells were examined under flow cytometry. Three independent experiments were performed in triplicates|
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|Figure 4: Effect of chelerythrine (CHE) on the expression of NKG2A on tumor associated NK (TANK) cells. Mice were administered with CHE (2.5 mg/kg) and staurosporine (1.0 mg/kg) after 24 (control)/34 (experimental) days of post DL cells transplantation, TANK cells were isolated using Magnetic Associated Cell Sorter (MACS-EasySep Magnet) and subjected to stained with specific anti-NKG2A-FITC antibody. Expression of NKG2A on TANK cells were examined under flow cytometry. Three independent experiments were performed in triplicates|
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| > Discussion|| |
In this study, we demonstrate that CHE delayed progressive growth of DL and increased survival duration of DL bearing BALB/c (H 2d ) mice. Further, results show that in vivo administration of CHE (2.5 mg/kg) results in increased cytotoxic function of TANK cells and recovery of immunosuppression caused by tumor invasion.
In corroboration with previous findings that demonstrated antitumor effect of CHE on HNSCC tumor model of SQ-20B xenograft in mice and a derivative of staurosporine, 7-hydroxystaurosporine (UCN-01) showed significant anticancer effect against breast cancer, B-chronic lymphocytic leukemia, but mechanisms of action was distinct from those identified for CHE. ,, This suggests that CHE showed a wide range of biological activities in vitro; however, very few reports available represented in vivo anticancer activities of CHE. ,,,, Here, we report that CHE delayed tumor growth and increased survival duration of DL bearing BALB/c (H 2d ) mice. Reduction in mean tumor volume was observed significant after administration of CHE (2.5 mg/kg) as compared to control as well as staurosporine treated mice.  The decrease in mean tumor volume was found superior after in vivo administration of cyclophosphamide (50 mg/kg). , Further, treatment results in increased survival duration of tumor bearing mice, which were observed consistent with previous findings. We also clarified the correlation between tumor regression and increased cytotoxic function of TANK cells after administration of CHE (2.5 mg/kg). [20-26] Results show that upregulated expression of activating receptor NKG2D on TANK cells increased cytotoxic function for tumor cells and enhanced recovery from immunosuppression caused by tumor growth. , Increased expression of NKG2D showed enhance cytotoxic function of TANK cells after in vivo administration of CHE (2.5 mg/kg). ,, Cyclophosphamide (50 mg/kg) produced better results than CHE.  It is widely known that NK cells are the connecting link between innate and cellmediated immune system therefore CHE might be an effective promoter of recovery achieved after immunosupression during tumor growth. This indicates that CHE stimulated both arms of the immune system to fight against tumor invasion. In contrast, expression of inhibitory receptor NKG2A on TANK cells was examined down regulated as compared to control. These results were coupled with a decrease in mean tumor volume and increased survival duration of BALB-c/H 2d mice treated with CHE (2.5 mg/kg). CHE delayed tumor growth and progression significantly as compared to staurosporine. Therefore, it can be used as a potential anticancer therapeutic agent. Regression in mean tumor volume might be results in induction of apoptosis as we reported in vitro. Further, reduction in tumor volume can be correlated with increased survival duration of tumor bearing mice. These findings suggest that apoptosis might be the predominant mechanism that causes reduction in mean tumor volume.  Similar results were observed in melanoma cells after treatment with staurosporine (1 μM) and its derivative UCN-01 in vitro.,, These results were correlated with cyclophosphamide (50 mg/kg), an established drug and being use in clinical practice were found consistent. Cyclophosphamide was used as a single agent for antitumor therapy based on the hypothesis that it normalizes immune competence of tumor bearing host.  Under the circumstances when early tumor was present CHE and cyclophosphamide delayed tumor growth significantly in BALB/c H 2d mice as compared to control and cyclophosphamide was found slight superior than CHE. It is possible that success of this comparative modality treatment is based on the ability of two agents to abrogate tumor mediated aberrations and regulation of immune response.  Such aberrations might occur in tumor bearing mice with excessive growth in mean tumor volume and marked decrease in survival duration of tumor bearing host. In other instances CHE, dependent change in DL bearing BALB/c (H 2d ) mice can lead a shift in tumor volume and increased survival duration of DL bearing BALB/c (H 2d ) mice. Therefore, the development of PKC inhibitor based antitumor therapy has interested in tumor immunology, and their remarkable example, NK 109, a CHE analogue, is in phase II trial in Japan and UCN-01 a PKC inhibitor, in phase I trial in UK. ,, Detailed investigation needed to explore the impact of novel insights into NK cell responses against tumors on the design of NK cell-based therapies. ,
| > Acknowledgment|| |
The project was supported by University Grants Commission (UGC), New Delhi. Mr. Sanjay Kumar expresses his appreciation to Indian Council of Medical Research (ICMR), New Delhi for student supports.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]