IN-VIVO EXPERIMENTATION OF TUPICHINOL E, OSIMERTINIB, AND THEIR COMBINATION USING A XENOGRAFT MODEL

IN-VIVO EXPERIMENTATION OF TUPICHINOL E, OSIMERTINIB, AND THEIR COMBINATION USING A XENOGRAFT MODEL

Adyasa Samantaray and Debasish Pradhan *

University Department of Pharmaceutical Sciences, Utkal University, Vani Vihar, Bhubaneswar, Odisha, India.

ABSTRACT: This comprehensive study investigates the in vivo effects of Tupichinol E, osimertinib, and their combination in a xenograft model of triple-negative breast cancer (TNBC), an aggressive and therapeutically challenging breast cancer subtype. By employing a multi-faceted approach, the study evaluates tumor growth inhibition, apoptosis, and proliferation through histopathological and immunohistochemical techniques, including hematoxylin and eosin (H&E) staining, TUNEL staining, and Ki67 immunohistochemistry. The results reveal significant therapeutic potential. Tumor growth was markedly suppressed in all treated groups, with monotherapy using Tupichinol E or Osimertinib reduces tumor volume by approximately 35% and 40%, respectively. Notably, combination therapy achieved a synergistic effect, maintaining tumor volumes at baseline levels and preventing progression over the two-week treatment period (p<0.001). Histological analyses demonstrated extensive necrosis and disruption of tumor architecture in the combination group, correlating with a 150% increase in apoptotic activity as indicated by TUNEL staining (p<0.001). Furthermore, Ki67 immunohistochemistry showed a substantial 70% reduction in proliferative activity in the combination group compared to controls (p< 0.001). These findings underscore the enhanced efficacy of combining Tupichinol E and Osimertinib, offering a novel and promising therapeutic strategy for TNBC. The synergistic effects observed in tumor suppression, apoptosis induction, and proliferation inhibition highlight the potential for this combination to address unmet clinical needs in TNBC treatment. This study provides a foundation for future research to explore molecular mechanisms and clinical applicability of this combination therapy.

Keywords: Tupichinol E, Osimertinib, Xenograft Model, H&E Staining, TUNEL Staining, and Ki67 Immunohistochemistry

INTRODUCTION: Triple-negative breast cancer (TNBC) is one of the most aggressive and clinically demanding types of breast cancer. TNBC, which accounts for 15-20% of all breast cancer cases, is distinguished by a lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2 amplification.

This unique molecular profile renders TNBC unresponsive to hormone therapies and HER2-targeted treatments, such as tamoxifen and trastuzumab that have significantly improved outcomes in other breast cancer subtypes. Consequently, chemotherapy remains the primary systemic treatment for TNBC, yet it often yields suboptimal results due to the high rates of recurrence, metastasis, and resistance ¹.

The clinical behavior of TNBC is marked by its aggressive nature, with higher rates of distant metastases to the lungs, brain, and liver. TNBC disproportionately affects younger women, and its prognosis remains poor, with a 5-year survival rate significantly lower than hormone receptor-positive breast cancers. The heterogeneous nature of TNBC at the molecular and cellular levels further complicates its management, as this diversity impacts treatment response and disease progression. Emerging molecular profiling techniques have begun to reveal actionable targets within subsets of TNBC, providing hope for more tailored and effective therapies ³.

One promising avenue for targeted therapy in TNBC is the epidermal growth factor receptor (EGFR) pathway. EGFR is overexpressed in approximately 50% of TNBC cases, making it a potential therapeutic target. Osimertinib, a third-generation EGFR inhibitor, has transformed the treatment of NSCLC with EGFR mutations. Osimertinib suppresses tumor cell proliferation, survival, and migration by selectively targeting the EGFR. Despite its success in NSCLC, the role of Osimertinib in TNBC remains underexplored, warranting investigation into its efficacy and mechanisms of action in this context ².

In parallel, there is growing interest in the use of natural compounds in cancer therapy. Tupichinol E, a novel bioactive compound derived from traditional medicinal plants, has shown potent anticancer activity in preclinical studies. Its actions include inducing apoptosis, inhibiting cell growth, and disrupting crucial signaling pathways involved in tumor formation. Unlike many synthetic chemotherapeutic agents, natural compounds like Tupichinol E often exhibits lower toxicity, making them attractive candidates for combination therapies ⁴.

The rationale for combining Osimertinib with Tupichinol E lies in their complementary mechanisms of action. While Osimertinib specifically targets EGFR-driven pathways, Tupichinol E exerts broader anticancer effects, including modulation of apoptosis and cell cycle regulators. Together, these agents have the potential to overcome the limitations of monotherapies, enhance therapeutic efficacy, and mitigate resistance mechanisms ⁵. This study aims to evaluate the therapeutic potential of Tupichinol E, Osimertinib, and their combination in a xenograft model of TNBC. Using advanced histological techniques, including hematoxylin and eosin (H&E) staining, TUNEL staining, and Ki67 immunohistochemistry, this research seeks to elucidate the effects of these treatments on tumor growth, apoptosis, and proliferation. By providing a deeper understanding of their mechanisms, the findings from this study could pave the way for the development of innovative therapeutic strategies for TNBC, addressing critical gaps in its treatment landscape. Furthermore, this research underscores the importance of integrating molecularly targeted therapies and natural compounds in the quest for effective and personalized cancer care.

MATERIALS & METHODOLOGY:

Reagents and Compounds: The in-vivo experimentation utilized a range of specialized reagents and components to ensure the precision and reliability of model ⁶. MDA-MB-231 cells, a human triple-negative breast cancer cell line, were selected to establish the xenograft tumors. These cells were suspended in phosphate-buffered saline (PBS) and mixed in a 1:1 ratio with Matrigel to enhance tumor engraftment and stability post-injection. Tupichinol E, a naturally derived bioactive compound, was prepared by dissolving it in saline containing 5% dimethyl sulfoxide (DMSO) for intraperitoneal administration ^{7, 9}. Osimertinib, a marketed third-generation EGFR inhibitor, was prepared and administered at a dose of 15 mg/kg intraperitoneally, once weekly, tailored for the murine model ²⁶.

In-vivo Experiment: Female BALB/c nude mice were procured from the National Institute of Animal Biotechnology (NIAB) in Hyderabad. The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee at The Pinnacle Biomedical Research Institute (PBRI) in Bhopal, under the protocol approval number PBRI/IAEC/21-03-2024/035. Upon arrival, the mice were individually housed in ventilated disposable cages, provided with food and water, and maintained under controlled environmental conditions (20 ℃, 50% ± 20% relative humidity, and a 12-hour light/dark cycle) ^{8, 10}. After a 7-day acclimatization period, each mouse was subcutaneously injected at the right flank with a suspension of 4 × 10⁶ MDA-MB-231 human breast cancer cells.

The cells were resuspended in phosphate-buffered saline (PBS), mixed in a 1:1 ratio with serum-free medium containing Matrigel. Tumor growth was monitored, and once the average tumor volume reached approximately 100 mm³ (calculated using the formula: Volume = ½ × width² × length), the mice were randomly assigned into four treatment groups (n=5 per group) as follows:

• Group 1 (Control): Saline water (intraperitoneal injection, 6 days per week).
• Group 2: Tupichinol E (5 mg/kg, intraperitoneal injection, 6 days per week).
• Group 3: Osimertinib (15 mg/kg, intraperitoneal injection, once per week).
• Group 4: Combination treatment with Tupichinol E (5 mg/kg, intraperitoneal injection, 6 days per week) and Osimertinib (15 mg/kg, intraperitoneal injection, once per week).

Both Tupichinol E and Osimertinib were dissolved in saline water containing 5% dimethyl sulfoxide (DMSO). Tumor volumes and body weights were measured twice weekly. After two weeks of treatment, the mice were euthanized. Tumor tissues were excised, weighed, and collected for histological and immunohistochemical analyses ¹¹.

Hematoxylin and Eosin (H&E) Staining: Tumor tissues were fixed in a 10% neutral buffered formalin solution (Sigma-Aldrich) before undergoing paraffin embedding. Thin sections of the tissues were prepared and subsequently stained using the H&E solution (Merck). The stained sections were then analyzed and imaged using a fluorescence microscope (Olympus BX51, Japan) ¹⁵.

Tunel Staining: Apoptotic activity in the tumor tissues was evaluated through TUNEL staining, following the manufacturer’s protocol. Briefly, the tissue sections were deparaffinized and rehydrated using a graded ethanol series. After rinsing with 0.85% sodium chloride (NaCl) and phosphate-buffered saline (PBS), the sections were fixed with 4% formaldehyde for 15 minutes. Proteinase K solution was applied to the tissue sections for 8–10 minutes following a PBS wash. A second fixation with 4% formaldehyde for 5 minutes was performed before equilibration in equilibrium buffer for 5–10 minutes. The TdT reaction mixture was then applied, and the sections were incubated for 1 hour in a dark environment. After this, the sections were treated with SSC solution for 15 minutes and washed with PBS. Finally, after counterstaining with DAPI, the sections were visualized and photographed under a fluorescence microscope (Olympus BX51, Japan). The average fluorescence signal from three images per treatment group was quantified ¹³.

Ki67 Immunohistochemistry: For Ki67 analysis, the tissue sections were first deparaffinized and subjected to antigen retrieval using citrate buffer. After blocking with 2% fetal bovine serum for 20–30 minutes, the sections were incubated with rabbit anti-human Ki67 antibody (1:200) for 1 hour at room temperature. Post-incubation, the sections were washed with PBS and treated with chicken anti-rabbit IgG TR-conjugated antibody (1:500) for 1 hour in a dark setting. The sections were counterstained with DAPI and subsequently imaged using a fluorescence microscope (Olympus BX51, Japan). Quantification of the fluorescence signals was performed by analyzing three images per treatment group ^{14, 13}.

Statistical Analysis: All experiments were conducted in triplicate, and the results were expressed as the mean ± standard deviation (SD). Statistical evaluations were performed using GraphPad Prism 9.0 software. Group comparisons were analyzed via one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant ^{8, 9}.

RESULT:

Tupichinol E Suppresses Breast Tumor Growth in Nude Mice: To establish a breast tumor xenograft model, MDA-MB-231 breast cancer cells were subcutaneously injected into the right flank of female nude mice. As illustrated in Fig. 1, the tumor volume in the vehicle-treated group exhibited a marked increase after two weeks, growing from approximately 100 mm³ to 330 mm³. Treatment with 5 mg/kg Tupichinol E and 15 mg/kg Osimertinib significantly slowed tumor progression; however, neither treatment completely eliminated the tumors or restored the tumor volume to baseline. Notably, the combination treatment (5 mg/kg Tupichinol E + 15 mg/kg Osimertinib) demonstrated a more pronounced inhibitory effect on tumor growth compared to either treatment alone. While the combination therapy did not completely eradicate the tumor, it successfully maintained tumor volume near the baseline level throughout the two-week treatment period ²¹. Additionally, a slight reduction in mouse body weight was observed across treatment groups, but no visible adverse effects were detected.

FIG. 1: TUPICHINOL E REDUCES BREAST TUMOR GROWTH IN NUDE MICE MDA-MB-231 CELLS WERE INJECTED SUBCUTANEOUSLY INTO THE RIGHT FLANK OF EACH NUDE MOUSE. Once tumors reached a volume of 100 mm³, the mice were randomly assigned into four groups (n=5/group). Group I received saline water (intraperitoneally, six days per week). Group II received 5 mg/kg Tupichinol E (intraperitoneally, six days per week). Group III received 15 mg/kg Osimertinib (intraperitoneally, six days per week). Group IV received a combination of 5 mg/kg Tupichinol E (intraperitoneally, once per week) and 15 mg/kg Osimertinib (intraperitoneally, six days per week). Tumor volumes are presented as means ± SD. *p<0.05, ***p<0.001 compared to the control group. **p<0.05 denotes comparison between indicated groups.

Histological and Immunohistochemical Analysis of Tumor Tissues: Tumor tissues were subjected to H&E staining to assess structural alterations ^{23, 24}. In the vehicle-treated group, high-grade tumors were observed with irregular cellular arrangements Fig. 2A. In contrast, tumors from drug-treated groups exhibited structural changes, characterized by increased cellular debris and reduced stromal content. TUNEL staining was performed to evaluate apoptosis levels in the tumor tissues. A comparison of green fluorescence signals revealed approximately 50% higher TUNEL staining in tumors treated with 5 mg/kg Tupichinol E or 15 mg/kg Osimertinib. Notably, the combined treatment group displayed a 150% increase in TUNEL staining compared to the vehicle group, indicating a synergistic enhancement in apoptotic activity Fig. 2B. Immunohistochemical analysis targeting the Ki67 protein, a well-established marker of cellular proliferation, showed significant reductions in Ki67 levels in all drug-treated groups relative to the vehicle-treated group. The lowest Ki67 expression was observed in the combined treatment group, highlighting the effectiveness of the combination therapy in suppressing tumor cell proliferation Fig. 2C.

FIG. 2: TUPICHINOL E ENHANCES APOPTOSIS AND REDUCES PROLIFERATION IN TUMOR TISSUES. (A) H&E staining revealed structural changes in tumor tissues across treatment groups. (B) TUNEL staining demonstrated enhanced apoptosis, with quantification based on the average number of green fluorescent dots per group. Values represent means ± SD from three images. *p<0.05, **p<0.01 compared to the vehicle group. (C) Ki67 immunohistochemical staining showed reduced proliferation, with quantification based on fluorescence signals. Values are means ± SD from three images. *p<0.05, **p<0.01 compared to the vehicle group.

DISCUSSION: The findings of this study underscore the potential of combining Tupichinol E and Osimertinib as a novel therapeutic strategy for triple-negative breast cancer (TNBC). The results demonstrated that this combination therapy offers superior tumor growth inhibition, enhanced apoptosis induction, and significant suppression of cell proliferation compared to either agent alone. These outcomes suggest that the combination of Tupichinol E and Osimertinib acts synergistically to target multiple pathways involved in TNBC progression ^{12, 19}.

One of the most striking findings was the ability of the combination therapy to maintain tumor volumes at baseline levels throughout the treatment period. This outcome highlights the potential of this approach to effectively control tumor progression in aggressive cancers like TNBC. The histological and immunohistochemical analyses further validated these results, with extensive necrosis and disruption of tumor architecture observed in the combination group. This degree of tumor cell death was corroborated by the significant increase in TUNEL staining, which indicated a 150% rise in apoptotic activity compared to the control group ¹⁵.

The reduction in Ki67 positivity in the combination group also provides critical insights into the mechanisms of action. Ki67, a marker of cellular proliferation, was significantly decreased by 70% in the combination therapy group. This reduction underscores the efficacy of this approach in curbing the rapid proliferation that is characteristic of TNBC cells. These findings align with the proposed mechanisms of Tupichinol E and Osimertinib, wherein the former induces apoptosis and modulates key signaling pathways, while the latter inhibits EGFR-driven proliferation and survival ¹⁶. The observed synergy between Tupichinol E and Osimertinib may be attributed to their complementary mechanisms of action.

While Osimertinib specifically targets EGFR, Tupichinol E has broader anticancer effects, including modulation of apoptosis-related proteins and inhibition of angiogenesis. This multi-pronged approach likely disrupts the ability of TNBC cells to adapt and survive under therapeutic pressure, thereby enhancing overall efficacy ²⁷.

In the context of existing therapies for TNBC, the results of this study are particularly significant. Current treatment options for TNBC are largely limited to chemotherapy, which is associated with high toxicity and limited efficacy in the face of drug resistance. The combination of Tupichinol E and Osimertinib presents a targeted, less toxic alternative that could improve patient outcomes. Moreover, the natural origin of Tupichinol E offers the added advantage of potentially reduced side effects, which is a critical consideration in cancer therapy ^{17, 21}.

Future research should focus on elucidating the molecular pathways underlying the observed synergy. For instance, investigating the effects of this combination on EGFR downstream signaling pathways, such as PI3K/AKT and MAPK, could provide deeper mechanistic insights. Additionally, exploring the role of apoptosis regulators like Bcl-2 and caspases could help clarify how Tupichinol E enhances the apoptotic effects of Osimertinib ²².

Clinical translation of these findings will require further validation in larger preclinical models and eventually in clinical trials. Optimizing the dosing regimen and identifying biomarkers for patient stratification will be crucial steps in this process. Given the heterogeneity of TNBC, understanding which subsets of patients are most likely to benefit from this combination therapy will be essential for its successful implementation ²⁵.

In conclusion, the combination of Tupichinol E and Osimertinib represents a promising therapeutic strategy for TNBC. By leveraging the complementary mechanisms of these agents, this approach addresses critical gaps in current treatment options, offering hope for improved outcomes in this challenging cancer subtype.

CONCLUSION: This study highlights the promising potential of combining Tupichinol E and Osimertinib as a therapeutic strategy for triple-negative breast cancer (TNBC), a cancer subtype with limited treatment options and poor prognosis. The results demonstrate that this combination therapy not only effectively inhibits tumor growth but also significantly enhances apoptosis and reduces cellular proliferation. These effects, driven by the complementary mechanisms of action of the two agents, underscore the potential of this approach to address the critical challenges posed by TNBC ¹⁸.

The ability of the combination therapy to maintain tumor volumes at baseline levels throughout the treatment period represents a significant advancement in the therapeutic management of aggressive cancers. By targeting both EGFR-driven pathways and broader tumor survival mechanisms, this approach effectively disrupts TNBC progression while potentially minimizing the toxicity associated with conventional therapies. Histological analyses revealing extensive necrosis and immunohistochemical findings of reduced Ki67 positivity provide strong evidence of the therapy’s efficacy ²⁰.

While these preclinical findings are highly encouraging, the path to clinical application involves addressing several key aspects. First, further studies should explore the molecular pathways involved in the observed synergy between Tupichinol E and Osimertinib. Understanding how these agents interact at a cellular and molecular level will facilitate the development of biomarkers to predict patient response. Second, larger preclinical studies should be conducted to validate these results and establish optimal dosing regimens. Third, the safety and efficacy of this combination therapy must be assessed in clinical trials, with an emphasis on identifying patient subsets most likely to benefit from this treatment ^{26, 28}.

In conclusion, the combination of Tupichinol E and Osimertinib represents a highly promising therapeutic approach for TNBC. By leveraging their complementary mechanisms of action, this strategy offers the potential for improved outcomes and reduced toxicity compared to current treatment options. This study provides a strong foundation for future research and highlights the importance of integrating natural compounds and molecularly targeted therapies in the fight against aggressive cancers like TNBC. Continued investigation and clinical validation of this approach could pave the way for a new paradigm in TNBC treatment, offering hope to patients facing this challenging disease.

ACKNOWLEDGMENT: This work is financially supported by research grants from the Bayers Foundation.

Data Availability Declaration: The authors confirm that the data supporting the findings of this study are available within the article. If any supporting data required, it is available from the corresponding author, upon reasonable request.

Author Contribution:

Adyasa Samantaray: Article Drafting, Analysis and interpretation of the data.

Debasish Pradhan: Supervision, Analysis, Conception and design.

CONFLICT OF INTEREST: No conflict of interest.

REFERENCES:

1. Burguin A, Diorio C & Durocher F: Breast Cancer Treatments: Updates and New Challenges. Journal of Personalized Medicine 2021; 11(8): 808. https://doi.org/10.3390/jpm11080808
2. Li Y, Zhang H and Merkher Y: Recent advances in therapeutic strategies for triple-negative breast cancer. J Hematol Oncol 2022; 15: 121. https://doi.org/10.1186/s13045-022-01341-0
3. Dass SA, Tan KL, Selva Rajan R, Mokhtar NF, Mohd Adzmi ER, Wan Abdul Rahman WF, Tengku Din TADAA & Balakrishnan V: Triple negative breast cancer: a review of present and future diagnostic modalities. Medicina 2021; 57(1): 62. https://doi.org/10.3390/medicina57010062
4. Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27-55. doi:10.1016/0065-2571(84)90007-4.
5. Chabanon RM, Rouanne M and Lord CJ: Targeting the DNA damage response in immuno-oncology: developments and opportunities. Nat Rev Cancer 2021; 21: 701–717. https://doi.org/10.1038/s41568-021-00386-6
6. Aaron C. Tan: Targeted therapies for lung cancer patients with oncogenic driver molecular alterations. JCO 2022; 40: 611-625. doi:10.1200/JCO.21.01626
7. Huang M, Lu JJ & Ding J: Natural Products in Cancer Therapy: Past, Present and Future. Natural Products and Bioprospecting 2021; 11(1): 5–13. https://doi.org/10.1007/s13659-020-00293-7
8. Wakefield L, Agarwal S and Tanner K: Preclinical models for drug discovery for metastatic disease. Cell 2023; 186(8): 1792-1813. doi: 10.1016/j.cell.2023.02.026
9. Adyasa Samantaray and Debasish Pradhan: Tupistra chinensis a traditional Chinese plant suppresses tumor growth and metastasis in-vitro against ER positive and triple negative breast cancer. Pharmacological Research - Modern Chinese Medicine 2025; 10: 100368. doi: https://doi.org/10.1016/j.prmcm.2024.100368
10. Duarte D & Vale N: Evaluation of synergism in drug combinations and reference models for future orientations in oncology. Current Research in Pharmacology and Drug Discovery 2022; 3: 100110. https://doi.org/10.1016/j.crphar.2022.100110
11. Zhu S, Wu Y and Song B: Recent advances in targeted strategies for triple-negative breast cancer. J Hematol Oncol 2023; 16: 100. https://doi.org/10.1186/s13045-023-01497-3
12. Louis DM, Nair LM, Vallonthaiel AG, Narmadha MP & Vijaykumar DK: Ki 67: a Promising Prognostic Marker in Early Breast Cancer-a Review Article. Indian Journal of Surgical Oncology 2023; 14(1): 122–127. https://doi.org/10.1007/s13193-022-01631-6
13. Mirzayans R and Murray D: Do TUNEL and Other Apoptosis Assays Detect Cell Death in Preclinical Studies? Int J Mol Sci 2020; 21(23): 9090. doi: 10.3390/ijms21239090. PMID: 33260475; PMCID: PMC7730366.
14. Bischoff H, Espié M & Petit T: Neoadjuvant Therapy: Current Landscape and Future Horizons for ER-Positive/HER2-Negative and Triple-Negative Early Breast Cancer. Curr. Treat. Options in Oncol 2024; 25: 1210–1224. https://doi.org/10.1007/s11864-024-01251-y
15. Udonkang M, Ene C, Archibong A, Egbe A & Inyang I: Aqueous beetroot dye as an alternative to haematoxylin and eosin in the diagnosis of breast tumours. Global Journal of Pure and Applied Sciences 2021; 27(4): 417-423.
16. Mandair D, Reis-Filho JS & Ashworth A: Biological insights and novel biomarker discovery through deep learning approaches in breast cancer histopathology. NPJ Breast Cancer 2023; 9(1): 21.
17. Yuan L, Cai Y, Zhang L, Liu S, Li P & Li X: Promoting apoptosis, a promising way to treat breast cancer with natural products: A comprehensive review. Frontiers in Pharmacology 2022; 12: 801662.
18. Laface C, Maselli FM, Santoro AN, Iaia ML, Ambrogio F, Laterza M & Fedele P: The resistance to EGFR-TKIs in non-small cell lung cancer: from molecular mechanisms to clinical application of new therapeutic strategies. Pharmaceutics 2023; 15(6): 1604.
19. Kulinowski Ł, Luca SV, Minceva M & Skalicka-Woźniak K: A review on the ethnobotany, phytochemistry, pharmacology and toxicology of butterbur species (Petasites L.). Journal of Ethnopharmacology 2022; 293: 115263. https://doi.org/10.1016/j.jep.2022.115263
20. Naeimzadeh Y, Tajbakhsh A, Nemati M & Fallahi J: Exploring the anti-cancer potential of SGLT2 inhibitors in breast cancer treatment in pre-clinical and clinical studies. European Journal of Pharmacology 2024; 176803.
21. Liang P, Ballou B, Lv X, Si W, Bruchez MP, Huang W & Dong X: Monotherapy and combination therapy using anti‐angiogenic nanoagents to fight cancer. Advanced Materials 2021; 33(15): 2005155.
22. Finkelman BS, Zhang H, Hicks DG & Turner BM: The evolution of Ki-67 and breast carcinoma: past observations, present directions, and future considerations. Cancers 2023; 15(3): 808.
23. Shaikh IA, Alshabi AM, Alkahtani SA, Orabi MA, Abdel-Wahab BA, Walbi IA & Hoskeri JH: Apoptotic cell death via activation of DNA degradation, caspase-3 activity, and suppression of Bcl-2 activity: an evidence-based Citrullus colocynthis cytotoxicity mechanism toward MCF-7 and A549 cancer cell lines. Separations 2022; 9(12): 411.
24. Wick MR: The hematoxylin and eosin stain in anatomic pathology-An often-neglected focus of quality assurance in the laboratory. Seminars in Diagnostic Pathology 2019; 36(5): 303–311. https://doi.org/10.1053/j.semdp.2019.06.003
25. Jung J: Human tumor xenograft models for preclinical assessment of anticancer drug development. Toxicological Research 2014; 30(1): 1–5. https://doi.org/10.5487/TR.2014.30.1.001
26. Zhang H: Osimertinib making a breakthrough in lung cancer targeted therapy. Onco Targets and Therapy 2016; 9: 5489–5493. https://doi.org/10.2147/OTT.S114722
27. Varghese R & Dalvi YB: Natural Products as Anticancer Agents. Current Drug Targets 2021; 22(11): 1272–1287. https://doi.org/10.2174/1389450121999201230204526
28. Jin L, Han W and Cao W: Synergistic Effects of Targeting EGFR and CDK4/6 in Preclinical Models of TNBC. Breast Cancer Res 2022; 24(1): 14.
    

    ---

    How to cite this article:

    Samantaray A and Pradhan D: In-vivo experimentation of Tupichinol E, osimertinib, and their combination using a xenograft model. Int J Pharm Sci & Res 2025; 16(7): 1959-66. doi: 10.13040/IJPSR.0975-8232.16(7).1959-66.

    ---

    All © 2025 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.