ABSTRACT
Background
Diospyros peregrina is dioecious plant native to India and belonging to the family of Ebenaceae, is largely
utilized in treatment of various ailments. Little has been known about the antitumor
activity of Diospyros peregrina with only 1 previous study on Ehrlich Ascites Carcinoma in mice. Therefore, it prompted
us to extensively explore the immunomodulatory effect in various cancer forms. The
focal point of this study revolves around breast cancer, which is the second most
common cancer in the world. In view of the increasing demands for noninvasive treatments,
natural plant-based agents open up promising applications in cancer immunotherapy
Methods
CD4+ lymphocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of
breast cancer patients and normal donor blood samples using magnetic-activated cell
sorting (MACS) and cultured separately. Utilizing the plastic surface adherence property,
the macrophages were isolated from CD4 negative lymphocytes of both breast cancer
patients and normal donors. For the presentation of tumor antigens invitro, macrophages
were pulsed with breast tumor associated antigen (BTAA) in presence or absence of
Diospyros peregrina fruit preparation (DFP). Differentially pulsed and irradiated macrophages were co-cultured
with autologous and allogenic lymphocytes. Supernatants hence collected from CD4+ lymphocytes were utilized for cytokine profiling using ELISA and proliferation was
assessed by MTT assay. Cytotoxic T lymphocytes (CTLs) generated from CD4 negative
lymphocytes culture (2 × 105) was incubated with MCF-7 (2 × 104) to check cytotoxicity using LDH release assay. CD4+ lymphocytes were treated in presence or absence of DFP, were analyzed using immunoblotting
and RT-qPCR, to check DFP mediated T helper (Th) cell differentiation through investigation
of signatory cytokines and transcription factors.
Results
It was found that DFP elevated the proliferation of CD4+ T lymphocytes (Th) in response to BTAA. DFP also helped in presenting BTAA pulsed
macrophages directing in the cytotoxic T-lymphocyte mediated immune response. Results
indicated that DFP preferentially highlighted Th1 commitment with type-1 specific
cytokines IFN-g and IL-12 and was indifferent in Th2 manifestation. DFP was not only
involved in the upregulation of Tbet mounted type-1 mediated immune response and activation
of STAT1 but also it downregulated STAT6 and GATA3, the functional activators and
regulators of type-2 immune response. Moreover, it was observed that DFP inhibited
the tumor-promoting environment modulated through Tregs by downregulating Foxp3 and
STAT5. Further, it was detected that DFP directs Th1 bias and results in attainment
of better suppression of breast tumor
Conclusion
The results collectively pointed out that DFP favored cell-mediated immune response
from BTAA antigen presentation on macrophages and also helping in the robust proliferation
of an entire spectrum of T helper lymphocytes which furthermore strengthen the underlying
immune responses, hence, fencing the body, of the progression of breast cancer.
Keywords
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Article info
Publication history
Published online: January 05, 2023
Accepted:
December 29,
2022
Received in revised form:
December 28,
2022
Received:
April 15,
2022
Identification
Copyright
© 2022 Elsevier Inc. All rights reserved.
