Effect on tumor volume, TIR, and survival rate

As illustrated in Fig. 1A, tumor volume increased gradually in the untreated SEC group in a time-dependent manner. At the end of the experiment, mice bearing SEC and treated with DOX, ATO, or LUT showed a significant (p < 0.05) decrease in tumor volume by 39.86%, 35.84%, and 30.66%, respectively, compared to the SEC untreated group (Table 2). Treatment with ATO + DOX, LUT + DOX, or ATO + LUT resulted in a significant (p < 0.05) reduction in tumor volume by 57.38%, 52.67%, and 66.30%, respectively, compared to the SEC group. The combined treatments demonstrated a significant decrease in tumor volume relative to the individual treatments. This effect was particularly notable in the ATO + LUT-free DOX treatment.

Fig. 1The alternative text for this image may have been generated using AI.

Effect of Various Treatments on (A) Tumor Volume, (B) Tumor Inhibition Rate, (C) Survival Rate (%) in Mice Bearing Solid Ehrlich Carcinoma (SEC). DOX: Doxorubicin (4 mg/kg i.p.), ATO: Atorvastatin (20 mg/kg i.p.), LUT: Luteolin (40 mg/kg i.p.). Data are presented as Mean ± SD (n=10), a: significant versus SEC, b: significant versus DOX, c: significant versus ATO, d: significant versus LUT, e: significant versus ATO+DOX, f: significant versus LUT+DOX.

Table 2 Effect of various treatments on tumor volume and weight

The calculated TIR further supported the antitumor efficacy of the treatment groups. Treatment with DOX, ATO, and LUT resulted in TIR of 39.81%, 38.80%, and 30.60%, respectively. Similarly, groups co-treated with ATO + DOX, LUT + DOX, and ATO + LUT exhibited TIR of 57.39%, 52.61%, and 67.28%, respectively. The TIR ranked in descending order: ATO + LUT > ATO + DOX > LUT + DOX > DOX > ATO > LUT (Fig. 1B).

To further evaluate these results, treatment interactions were assessed using observed and expected FTV. The expected FTV values were 0.434 for ATO + DOX, 0.478 for ATO + LUT, and 0.461 for LUT + DOX, while the corresponding observed FTV values were 0.420, 0.342, and 0.460, respectively. Calculation of the synergistic ratio (R) revealed that the ATO + DOX and ATO + LUT combinations exhibited synergistic effects, with R values of 1.03 and 1.40, respectively. In contrast, the LUT + DOX combination exhibited an additive interaction (R = 1.00). Collectively, these findings indicate that the co-treated groups demonstrated significant inhibition of tumor growth compared with the respective individual treatment groups.

The survival rates during the experimental period were also examined. The SEC control group demonstrated a 71.4% survival rate, while the DOX-treated group had a survival rate of 78.6%. Individual treatments with ATO or LUT showed slightly higher survival rates, with 85.7%. In contrast, combination treatments showed the highest survival rates with 92.9% in ATO + DOX, LUT +DOX, and ATO + LUT groups (Fig. 1C).

Effect on tumor weight

As illustrated in Table 2, all treatments significantly (p < 0.05) decreased tumor weight compared to the SEC control group. Treatment with DOX, ATO, or LUT groups resulted in a significant (p < 0.05) reduction in tumor weight by 39.18%, 38.43%, and 37.25%, respectively, compared to the SEC group. Similarly, treatment with ATO + LUT, ATO + DOX, and LUT + DOX significantly (p < 0.05) reduced tumor weight by 74.69%, 65.96%, and 57.53%, respectively, compared to the SEC group. Combined treatments, especially ATO +LUT, significantly reduced tumor weight compared to all treatment groups (Table 2).

Effect on ABCB1 and ABCG2 gene expression

Treatment of tumor tissue significantly suppressed ABCB1 and ABCG2 gene expression, as illustrated in Fig. 2. Mice treated with DOX, ATO, or LUT demonstrated a significant (p < 0.05) downregulation in ABCB1 expression by 5.7%, 17.1%, and 13.5%, and in ABCG2 expression by 22.4%, 10.3%, and 15.6%, respectively, relative to the SEC untreated group.

Fig. 2The alternative text for this image may have been generated using AI.

Effect of Various Treatments on (A) ABCB1 Gene Expression and (B) ABCG2 Gene Expression in Mice Bearing Solid Ehrlich Carcinoma (SEC). DOX: Doxorubicin (4 mg/kg i.p.), ATO: Atorvastatin (20 mg/kg i.p.), LUT: Luteolin (40 mg/kg i.p.). Data are presented as Mean ± SD (n=10), a: significant versus SEC, b: significant versus DOX, c: significant versus ATO, d: significant versus LUT, e: significant versus ATO+DOX, f: significant versus LUT+DOX. ABCB1: ATP-binding cassette (ABC) subfamily B member 1, ABCG2: ABC subfamily G member 2.

Similarly, co-treatment with ATO + DOX, LUT + DOX, and ATO + LUT exerted a significant (p < 0.05) reduction in ABCB1 expression by 59.1%, 43.4%, and 54.8%, and in ABCG2 expression by 53.8%, 31.4%, and 40.4%, respectively, compared to the SEC untreated group. Among all treated groups, the co-treated with ATO + DOX exhibited the most significant reduction in ABCB1 and ABCG2 gene expression (Fig. 2).

Effect on TERT and P-TERT protein expression

As shown in Fig. 3, mice bearing SEC and treated with DOX, ATO, or LUT showed a significant (p < 0.05) reduction in TERT protein expression by 14.88%, 19.28%, and 28.66%, respectively, compared to the SEC group. However, no significant difference in TERT protein expression was observed between the individual treatments of DOX and ATO. On the other hand, the groups co-treated with ATO + DOX, LUT + DOX, and ATO + LUT showed a significant (p < 0.05) reduction in TERT protein expression by 34.32%, 62.8%, and 70.5%, respectively, compared to the SEC group (Fig. 3A, B).

Fig. 3The alternative text for this image may have been generated using AI.

Effect of Various Treatments on Protein Expression of TERT and P-TERT: (A)Representative Western Blot Bands of TERT (~127 kDa), P-TERT (~127 kDa), and β-actin (~44kDa), ( B) Relative Protein Expression of TERT/ β-actin, (C) Relative Protein Expression of P-TERT/β-actin, (D) TERT/P-TERT Ratio. DOX: Doxorubicin (4 mg/kg i.p.), ATO: Atorvastatin (20mg/kg i.p.), LUT: Luteolin (40 mg/kg i.p.), SEC: Solid Ehrlich carcinoma. Data are presented asMean±SD (n=3), a: significant versus SEC, b: significant versus DOX, c: significant versus ATO, d:significant versus LUT, e: significant versus ATO+DOX, f: significant versus LUT+DOX. TERT:Telomerase reverse transcriptase, P-TERT: Phosphorylated telomerase reverse transcriptase.

Additionally, the mice bearing SEC treated with DOX, ATO, or LUT showed a significant (p < 0.05) reduction in P-TERT protein expression by 9.7%, 23.01%, and 19.57%, respectively, compared to the SEC-untreated group. Similarly, ATO + DOX, LUT + DOX, or ATO + LUT co-treated groups had a significant (p < 0.05) reduction in P-TERT protein expression by 39.74%, 52.39%, and 68.67%, respectively, compared to the SEC group. Co-treated groups demonstrated a significant decrease in TERT and its phosphorylated form protein expression relative to individual treatment groups (Fig. 3A, C). Notably, ATO + LUT combined treatment showed the most significant reduction in both TERT and P-TERT compared to all other treatments (Fig. 3).

To further assess the effect of these treatments, we calculated the TERT/P-TERT ratio in each group to evaluate changes in TERT phosphorylation status. A lower ratio reflects enhanced TERT phosphorylation, whereas a higher ratio indicates reduced phosphorylation. DOX, LUT, and LUT + DOX treatments resulted in a decreased ratio, indicating increased TERT phosphorylation. In contrast, ATO, ATO + DOX, and ATO + LUT increased the ratio, suggesting suppression of TERT phosphorylation (Fig. 3D).

Effect on histopathology of solid tumor tissue

As illustrated in Fig. 4(A-C), in the control SEC group, histopathological examination revealed viable foci with densely packed, round, polygonal, large, deeply stained tumor cells interspersed with small eosinophilic necrotic zones, which replaced the subcutaneous and muscular tissues. Furthermore, the SEC group showed a high number of unusual mitotic figures (3.5 ± 0.52), tumor giant cells (2.6 ± 0.52), and minimal nuclear karyorrhexis (0.7 ± 0.48), with a small number of apoptotic cells (2.4 ± 0.52) (Fig. 7).

Fig. 4The alternative text for this image may have been generated using AI.

Representative photomicrographs of H&E-stained solid tumor sections of the SEC and DOX groups. (A-C) SEC control group: A showing viable foci consisting of densely packed large, round, and polygonal deeply stained tumor cells (black arrows) interspaced by small eosinophilic necrotic zones (white arrows) replacing subcutaneous and muscular tissues (magnification ×100: scale bar 100 μm). B & C showing numerous atypical mitotic figures (yellow arrowheads), tumor giant cells (open arrowheads), and very mild nuclear karyorrhexis (blue arrows), as well as a few apoptotic cells (curved arrow) (magnification ×400; scale bar 50 μm). (D-F) DOX group: D showing smaller viable foci containing shrunken tumor cells (black arrows) interspaced by larger eosinophilic necrotic zones (white arrows) than in the SEC group (magnification ×100; scale bar 100 μm). E & F showing very few atypical mitotic figures (yellow arrowheads), very few tumor giant cells (open arrowheads), and increased nuclear karyorrhexis (blue arrows) were observed with many apoptotic cells (curved arrow) (magnification ×400; scale bar 50 μm).

Fig. 5The alternative text for this image may have been generated using AI.

Representative photomicrographs of H & E-stained solid tumor sections of ATO and LUT groups. (A-C) ATO group: A showing smaller viable foci containing shrunken tumor cells (black arrows) interspaced by large eosinophilic necrotic zones (white arrows) (magnification×100; scale bar 100 μm). B & C showing very few tumor giant cells (open arrowheads), increased nuclear karyorrhexis (blue arrows), and many apoptotic cells (curved arrow) (magnification×400; scale bar 50 μm). (D-F) LUT group: D showing many viable foci containing shrunken tumor cells (black arrows) interspaced by eosinophilic necrotic zones (white arrows) (magnification ×100; scale bar 100 μm). E & F showing few tumor giant cells (open arrowheads), prominent nuclear karyorhexis (blue arrows), and many apoptotic cells (curved arrow) (magnification ×400; scale bar 50 μm).

As shown in Fig. 4(D-F), sections from mice treated with DOX showed smaller viable foci with reduced tumor cells than the SEC untreated group. The DOX-treated group exhibited a significant (p < 0.05) reduction in atypical mitotic figures by 85.7% (0.5 ± 0.52) and tumor giant cells by 84.6% (0.4 ± 0.516), with elevated nuclear karyorrhexis by 142.85% (1.7 ± 0.48) and apoptotic cells by 187.5% (6.9 ± 0.99) compared to the SEC untreated group (Fig. 7).

Tumor sections from mice treated with ATO revealed fewer viable foci with reduced tumor cells than the SEC untreated group (Fig. 5A-C). Sections of the ATO group also demonstrated a significant (p < 0.05) reduction in tumor giant cells by 88.5% (0.3 ± 0.48) with a substantial increase in nuclear karyorrhexis by 157.14% (1.8 ± 0.42) and apoptotic cells by 145.8% (5.9 ± 0.73) compared to the SEC group (Fig. 7).

Fig. 6The alternative text for this image may have been generated using AI.

Representative photomicrographs of H&E-stained solid tumor sections of co-treatedgroups. (A, B) ATO+DOX group; A showing few smaller viable foci containing shrunkentumor cells (black arrows) interspaced by widespread eosinophilic necrotic zones (whitearrows) (magnification ×100; scale bar 100 μm). B showing few tumor cells within theviable areas, very few tumor giant cells (open arrowheads), prominent nuclear karyorrhexis(blue arrows), and many apoptotic cells (curved arrow) (magnification ×400; scale bar50 μm). (C, D) LUT+DOX group: C showing viable foci containing shrunken tumor cells(black arrows) interspaced by eosinophilic necrotic zones (white arrows) (magnification×100; scale bar 100 μm). D showing few tumor giant cells (open arrowheads), very fewatypical mitotic figures (yellow arrowheads), prominent nuclear karyorrhexis (blue arrows),and many apoptotic cells (curved arrow) were observed (magnification X400: bar 50). (E,F) ATO+LUT group; E showing small viable foci containing few shrunken tumor cells(black arrows) interspaced by eosinophilic necrotic zones (white arrows) (magnification×100; scale bar 100 μm). F showing very few tumor giant cells (open arrowheads), nomitotic figures, marked nuclear karyorrhexis (blue arrows), and many apoptotic cells(curved arrow) (magnification ×400; scale bar 50 μm).

Moreover, sections from mice treated with LUT demonstrated multiple viable foci with shrunken tumor cells (Fig. 5D-F). LUT treatment revealed a significant decrease in tumor giant cells by 53.8% (1.2 ± 0.42) and an increase in nuclear karyorrhexis by 157.14% (1.8 ± 0.42) and apoptotic cells by 175% (6.6 ± 0.52) compared to the SEC control group (Fig. 7).

Tumor sections from the ATO + DOX group revealed a few smaller viable foci containing shrunken tumor cells, as well as a few tumor cells within the viable areas of the tumor (Fig. 6A, B). ATO+DOX treatment showed a significant reduction in tumor giant cells by 92.3% (0.2 ± 0.42), an increase in nuclear karyorrhexis by 314.3% (2.9 ± 0.32), and an elevation in the apoptotic cells by 450% (13.2 ± 0.79) compared to the control SEC group (Fig. 7).

Fig. 7The alternative text for this image may have been generated using AI.

The Effect of Various Treatments on Tumor Histopathology; (A) Number of apoptotic cells, (B) Number of nuclear karyorrhexis, (C) Number of nuclei having mitotic figures, and (D) Number of tumor giant cells. DOX: Doxorubicin (4 mg/kg i.p.), ATO: Atorvastatin (20 mg/kg i.p.), LUT: Luteolin (40 mg/kg i.p.). SEC: Solid Ehrlich carcinoma. Data are presented as Mean ± SD (n=10). a: significant versus SEC, b: significant versus DOX, c: significant versus ATO, d: significant versus LUT, e: significant versus ATO+DOX, f: significant versus LUT+DOX. HPF: High power field.

Tumor sections from mice receiving LUT+ DOX treatment showed viable foci containing shrunken tumor cells (Fig. 6C, D). LUT + DOX treatment also showed a significant decrease in tumor giant cells by 88.5% (0.3 ± 0.48), a reduction in mitotic figures by 85.7% (0.4 ± 0.52), an increase in nuclear karyorrhexis by 271.4% (2.6 ± 0.51), and an increase in apoptotic cells by 387.5% (11.7 ± 1.16) compared to the control SEC group (Fig. 7).

Fig. 6(E, F) showed that tumor sections from mice bearing SEC and treated with ATO + LUT revealed small viable foci containing a few shrunken tumor cells. Sections from the ATO + LUT-treated group also showed a significant reduction of tumor giant cells (0.2 ± 0.41) by 92.3% relative to the SCE group. There was also a significant increase in nuclear karyorrhexis (2.9 ± 0.31) and apoptotic cells (15.8 ± 0.79) by 314.3% and 558.3%, respectively, compared to the SEC group (Fig. 7).

The co-treated groups, especially the ATO + LUT group, exhibited a significant (p < 0.05) increase in apoptotic cells compared to all treatments and nuclear karyorrhexis compared to individual treatment groups (Fig. 7A, B). Mitotic figures were observed in the SEC, DOX, and LUT + DOX groups but were absent in the ATO, LUT, ATO + DOX, and ATO + LUT-treated groups (Fig. 7C). All treatments revealed a significant reduction in tumor giant cells relative to the SEC control group (Fig. 7D).

Immunohistochemical localization of CD44 in solid tumor tissue

Fig. 8(A–G) illustrates the immunohistochemical staining of tumor sections from the control and treated groups. The SEC control group exhibited strong positive CD44 expression (90.35 ± 0.86). Tumor sections from the DOX group showed strong CD44 immunostaining, but this was significantly reduced by 12% (79.5 ± 0.8) compared to the SEC group (P < 0.05). ATO or LUT individual treatments resulted in moderate CD44 expression, with significant reduction by 48.78% (46.28 ± 1.49) and 64.89% (31.72 ± 1.29), respectively, compared with the SEC group. Notably, combined treatments with ATO + DOX, LUT + DOX, and ATO + LUT exhibited weak CD44 immunostaining, with significant reductions by 76.24% (21.47 ± 1.49), 79.07% (18.91 ± 0.91), and 86.73% (11.99 ± 0.99), respectively, compared to the SEC group. Among all treatments, the ATO + LUT combined treatment demonstrated the most significant reduction in CD44 expression. Furthermore, all combined treatments significantly decreased CD44 expression compared with the corresponding individual treatment groups, as illustrated in Fig. 8H.

Fig. 8The alternative text for this image may have been generated using AI.

Microscopic pictures of immunostained sections from solid Ehrlich carcinoma (SEC) tumors against CD44. Immunostaining showed strong positive expression in SEC control group (A) and DOX group (B), moderate expression in ATO (C) and LUT (D) groups, and weak expression in ATO+DOX (E), LUT+DOX (F), and ATO+LUT (G) groups (magnification ×400; scale bar 50µm). H: The Percent (%) Expression of CD44 immunostained sections in various treated groups: Data are presented as Mean±SD (n=10), a: significant versus SEC, b: significant versus DOX, c:significant versus ATO, d: significant versus LUT, e: significant versus ATO+DOX, f: significant versus LUT+DOX. DOX: Doxorubicin, ATO: Atorvastatin, LUT: Luteolin.