**1. Introduction**

Of the three classifications of macroalgae, the chemistry of red algae is more diverse than that of green or brown algae. Therefore, red algae are considered the most important source of many biologically active metabolites in comparison with other algal classes [1,2]. The genus *Hypnea* is one of the widest spread red algae, with economic importance as a source of carrageenan [3]. The *Hypnea* species were extensively assessed for their biological activities. Methanolic extract of *Hypnea flagelliformis* was subjected to the 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical scavenging assay, and it showed a stronger antioxidant activity compared with the standard quercetin [4]. Likewise, the ethyl acetate fraction showed a significantly higher total phenolic content, DPPH·scavenging activity, H2O2 scavenging activity, and lipid peroxidation inhibition than its crude extract, fractions of *n*-hexane and dichloromethane, and other methanolic fractions of *Hypnea valentiae*. This study introduced the red seaweeds *Hypnea* sp. to be used as food supplements for increasing shelf-life in the food industry and combating carcinogenesis [5]. Methanol extract of *Hypnea valentiae* inhibited acetylcholinesterase (AChE), and this neuroprotective action is considered a first line in the treatment of dementia [6]. Bitencourt and his colleagues observed that a lectin isolated from the red marine alga *Hypnea cervicornis* possessed antinociceptive and anti-inflammatory activity via interaction with the lectin carbohydratebinding site. Additionally, the lectin did not show visible signs of toxicity at effective doses [7]. The methanolic extract of the algea *Hypnea esperi* from the Suez Canal region showed potent antibacterial activity toward Gram-positive bacteria that correlated to long chain fatty acids of more than 10 carbon atoms in length, which induced lysis of bacterial protoplasts. In addition, *H. esperi* also exhibited anticoagulant activity by delaying the blood clotting to 120 s in comparison with the control blood's 40-s clotting time [8]. Moreover, several fatty acids such as palmitic, oleic, pentacosanoic, and hexacosenoic acids, as well as sesquiterpene and sterols, were reported in *Hypnea* [9]. The previously isolated compounds from the genus *Hypnea* can be classified into three categories; sterols and ketosteroids, terpenoids, and polymers as polypeptides and polysaccharides [10–15]. Although many biological studies were performed, less research work was performed for the isolation of these pure active compounds. Our study was oriented toward finding out other classes of bioactive compounds that attributed to the previously mentioned pharmacological activities of *Hypnea* sp.

The sphingolipid-signaling pathway is a novel anticancer target system. It has been suggested that sphingolipids play fundamental roles in the regulation of cancer pathogenesis and development [16]. Ceramide serves as a central mediator in sphingolipid metabolism and signaling pathways, regulating many essential cellular responses [17]. Many drugs used in the treatment of cancer are themselves ceramide generators. This property contributes in part to their apoptosis-inducing effects [18]. Consequently, targeting the ceramide-signaling pathway by activating ceramide downstream receptors, inhibiting ceramide-metabolizing enzymes, or exogenously increasing the ceramide levels comprise the novel targets for cancer treatment [19]. In the current work, we aimed to assess the potential antitumor and apoptotic activities of two novel ceramides isolated from *Hypnea musciformis*.

#### **2. Results and Discussion**

#### *2.1. Metabolic Analysis Profile*

The metabolic analysis profile produced by the LC-HR-ESI-MS technique (Figures S1 and S2) manifested different metabolites (Table 1 and Figure 1) that were detected by comparing their detected masses with those recorded in some databases (e.g., the Dictionary of Natural Products (DNP) and Metabolite and Chemical Entity (METLIN)). The mass accuracy was calculated by ((detected mass − expected mass)/expected mass) × 10<sup>6</sup> and expressed in parts per million (ppm) error [20]. Sterols with cholesterol nucleui were the most common chemical class isolated from *Hypnea musciformis*. Other bioactive metabolites were identified as ptilodene, an antimicrobial and anti-inflammatory icosanoid [21], agardhilactone, an oxylipin with epoxide ring showing anticancer activity [22,23], and oxytocic prostaglandin-E2, which induces labor [24]. Additionally, a phytosphingosine base that exhibited antiphlogistic and antimicrobial activity against Gram-positive and Gram-negative bacteria, viruses, and fungi [25,26] was detected. Therefore, the above-mentioned biological activity may have been related to the identified metabolites.

**Table 1.** Metabolic profiling (LC-ESI-HRMS) of methanolic crude extract of *Hypnea* sp.


**Figure 1.** Structures of the identified metabolites listed in Table 1.

#### *2.2. Identification of Isolated Compounds*

Ceramide **A** (**1**) (Figure 2) was obtained as a white powder, and its molecular formula was determined to be C43H83NNaO4 by ESI-HRMS with *m*/*z* 700.5921 [M + Na]+ (Figure S3), calculated as 700.6220, representing 3 degrees of unsaturation. The 1H NMR and 13C NMR spectral data of ceramide **A** (**1**) are listed in Table 2 (Figures S4 and S5). The backbone of a ceramide nucleus was recognized by the presence of an amide group at *δ*H 7.26 ppm/*δ*C 175.74 ppm and multiplet peaks of a long methylene chain at *δ*H 1.12–1.32 ppm/*δ*C 29.2–29.7 ppm. An oxygenated methylene was determined at *δ*H 3.75, 4.08 ppm/*δ*C 61.9 ppm. Two oxygenated methine groups as well as a nitrogen-bearing methine were determined at *δ*H 4.08 ppm/*δ*C 74 ppm, *δ*H 4.23 ppm/*δ*C = 72.5 ppm, and *δ*H 3.9 ppm/*δ*C 54.4 ppm, respectively. Four olefinic peaks were detected at *δ*C 129, 134.1, 123.1, and 136.3 ppm and *δ*H 5.51, 5.67, and 5.08 ppm. The length of the fatty acid chain was analyzed by HRMS after performing a protocol of methanolysis [27]. The HRMS showed a molecular ion peak at *m*/*z* 369.3231 [M + H]+ (Figure S6), calculated as *m*/*z* 369.3290, indicating a C22 fatty acid with a molecular formula of C23H45O3, recognized as 2-hydroxy docosanoic acid methyl ester, while the long chain base was recognized as 1,3-dihydroxy-2-amino-9-methyl-icosene-4,8-diene. Straight chains of both the fatty acid and sphingosine base ended with terminal methyl groups of a normal form at *δ*H 0.88 ppm/*δ*C 14 ppm. Finally, ceramide **A** (**1**) could be identified as a ceramide with alphahydroxylated unsaturated fatty acid and long chain of 9-methyl-sphinga-4,8-diene base possessing 2*S*, 2*R*, 3*R* relative configurations. The configuration of the ceramide moieties was assigned by comparing its physical data, optical rotation [α]22D +6.7 *c* 0.23, CHCl3), 1H-NMR, and 13C-NMR (measured in CDCl3) with analogs, using deuterated chloroform as an NMR solvent as reported in the literature [28,29]. The structure of compound **1** was determined to be <sup>2</sup>-hydroxy-N-[(2*S*,2*R*,3*R*,4*E*,8*<sup>E</sup>*)-1,3-dihydroxy-9-methyl-icosene-4,8- diene-2-yl]-docosanamide which, to the best of our knowledge, is a new compound.

**Figure 2.** Chemical structures of isolated compounds **1**–**6**.

Ceramide **B** (**2**) (Figure 2) was obtained as a white powder, and its molecular formula was determined to be C36H72NO5 by ESI-HRMS with *m*/*z* 584.4410 [M + H]+ (Figure S7), calculated as 584.5176, representing two degrees of unsaturation. The 1H NMR and 13C NMR spectral data are listed in Table 2 (Figures S8 and S9). The backbone structure of compound **2** was identified as a ceramide. The core of a ceramide nucleus was confirmed by the presence of an amide group at *δ*C 175.1 ppm/*δ*H 8.58 ppm and an overlapped long methylene chain at *δ*C 29.6 ppm/*δ*H 1.22–1.29 ppm. An oxygenated methylene group as well as a nitrogen-bearing methine group were determined at *δ*H 4.40, 4.5 ppm/*δ*C 61.7 ppm and *δ*H 5.11 ppm/*δ*C 52.6 ppm, respectively. Three groups of oxygenated methine were detected at *δ*C 72.1, 72.7, and 76.4 ppm and *δ*H 4.61, 4.28, and 4.35 ppm. Additionally, two olefinic peaks were determined at *δ*H 5.47 ppm/*δ*C 130.0 ppm. Terminal methyl groups of a normal type were detected at *δ*C 13.9 ppm/*δ*H 0.85 ppm. The length of the fatty acid chain was determined on the basis of the results of its oxidative methanolysis followed by peak detection by HRMS, which showed a molecular ion peak at *m*/*z* 313.2709 [M + H]+ (Figure S10), calculated as *m*/*z* 313.2743, indicating a methyl ester of C18:1 fatty acid with a molecular formula of C19H37O3. Therefore, the fatty ester methyl ester moiety was recognized as 2-hydroxy-10-nonadecenoic acid methyl ester, which was confirmed by GC-MS analysis (Figure S11). At last, ceramide **B** (**2**) could be identified as a ceramide with 2-hydroxy monounsaturated fatty acid and a long chain phytosphingosine base possessing 2*S*,2*R*,3*S*,4*R,*9*Z* relative configurations. The configuration of the ceramide moieties was assigned by comparing its physical data, optical rotation [α]22D +7.70 (*c* 0.27, pyridine), 1H-NMR, and 13C-NMR (measured in C5D5N) with the analogs using deuterated pyridine as an NMR solvent, as reported in the literature [27]. The structure of ceramide **B** (**2**) was determined to be <sup>2</sup>-hydroxy-N-[(2*S*,2*R,*3*S*,4*R,*9*Z*)-1,3,4-trihydroxy-nonadecan-2-yl]-10- heptadecenamide which, to the best of our knowledge, is a new compound.


**Table 2.** The 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of isolated compounds **1**, **2**, and **3** (*δ* in ppm, *J* in Hz).

Ceramide **C** (**3**) (Figure 2) was obtained as a white powder, and its molecular formula was determined to be C34 H70NO4 by ESI-HRMS. The mass spectrum of compound **3** displayed a molecular ion peak with *m*/*z* 556.5660 [M + H]+ (Figure S12), calculated as 556.5305, representing one degree of unsaturation. The 1H NMR and 13C NMR spectral data of ceramide **C** (**3**) are listed in Table 2 (Figures S13 and S14). The backbone structure of compound **3** was determined to be a ceramide, as explained above. In addition, the length of the fatty acid was analyzed by HRMS after its methanolysis. The HRMS showed a molecular ion peak at *m*/*z* 271.9316 [M + H]+ (Figure S15), calculated as *m*/*z* 271.2637, indicating a C16 fatty acid with a molecular formula of C17 H35 O3. Therefore, the fatty acid methyl ester moiety was recognized as a palmitic acid methyl ester. Consequently, ceramide **C** (**3**) can be identified as a ceramide with non-hydroxylated saturated fatty acid and a long chain phytosphingosine base possessing 2*S*,3*S*,4 *R* relative configurations. The structure of ceramide **C** (**3**) was determined to be N-[(2*S*,3 *R*,4 *R*)-1,3,4-trihydroxy-octadecan-2-yl] hexadecanamide. The configuration of the ceramide moieties was assigned by comparing its physical data, optical rotation [α]<sup>22</sup> D +14.30 (*c* 0.25, pyridine), and 1H and 13C NMR data with the reported data in the literature.

It was found that it was previously isolated under the name (2*S*,3*S*,4 *R*)-2-N-(palmitoyl)- phytosphingosine from *Armillaria mellea* [30]. However, it is worth mentioning that ceramide **3** is the first report of this ceramide in *Hypnea musciformis*. Furthermore, it was denoted as ceramide **C** in our biological study.

Other known compounds **4**–**6** (Figure 2) were identified as docosanoic acid (**4**) (Figure S16), hexadecanoic acid (**5**) (Figure S17), and alpha hydroxy octadecanoic acid (**6**) (Figure S18) by comparing the NMR data with the literature [30].

#### *2.3. In Vitro Cytotoxic Activity of Isolated Ceramides* **A** (**1**)*,* **B** (**2**)*, and* **C** (**3**)

The anticancer activity of ceramides was previously reported against different malignant cell lines [31–35]. The selection of the human breast adenocarcinoma (MCF-7) cell line was based on the global prevalence of breast cancer as well as common side effects of anticancer drugs that may be relatively ineffective against some phases [36]. From Table 3, it was noticed that ceramides **A** (**1**) and **B** (**2**) showed higher in vitro cytotoxic activity than ceramide **C** (**3**) against the MCF-7 cell line. Both ceramides **A** (**1**) and **B** (**2**) exhibited a promising in vitro cytotoxic activity with IC50 of 11.07 ± 0.23 μM and 10.17 ± 0.15 μM, respectively, when compared with doxorubicin as a positive control with IC50 of 8.65 ± 0.03 μM. The weak in vitro cytotoxic activity of ceramide **C** could be attributed to the absence of an olefinic group and hydroxy fatty acid, in addition to a fatty acyl chain of a shorter length.

**Table 3.** IC50 values (μM) of ceramides **A** (**1**), **B** (**2**), and **C** (**3**) against human breast cancer MCF-7 cell line using doxorubicin as a positive control.


\* Significantly different compared with positive control doxorubicin. Each data point represents the mean ± SD of three independent experiments (significant differences at *p* < 0.05).

The 2-hydroxy fatty acid, like the 2-hydroxy oleic acid, possessed antitumor activity against several types of cancer. Aside from this, the hydroxylation of fatty acids at C2 made some cancer cells sensitive to the antitumor drug [37]. Therefore, further assessment of the in vivo cytotoxic activity of ceramides **A** (**1**) and **B** (**2**) was performed.

#### *2.4. The Antitumor Effects of Isolated Ceramides* **A** (**1**) *and* **B** (**2**) *in a Mouse Model of Ehrlich Ascites Carcinoma (EAC)*

#### 2.4.1. Effect of the Investigated Ceramides on Liver and Kidney Function Markers

The serum levels of the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the kidney markers urea and creatinine, were determined in all the study groups to assess the possible toxicity of the investigated ceramides in the liver and kidneys. The results showed slightly higher levels of kidney and liver markers in the EAC control group and all the treated groups in comparison with the normal group but with no significant differences, indicating that the investigated doses of ceramide **A** (**1**) and ceramide **B** (**2**) had no detected toxicity in either the liver or kidneys (Table S1). No other toxic effects were detected in the experimental mice. There were also no observed changes in the behavior of the treated mice nor a marked increase in their mortality rates.

#### 2.4.2. Effect of the Investigated Ceramides on the Tumor Weight

In accordance with the findings of the in vitro study, all groups treated with either ceramide **A** (**1**) or ceramide **B** (**2**) showed a significant decrease in tumor weight compared with the Ehrlich ascites carcinoma (EAC) control group (*p* < 0.001) (Figure 3).

**Figure 3.** Effect of treatment with ceramide **A** and ceramide **B** at two dose levels (1 mg/kg and 2 mg/kg I.P) on the tumor weight in EAC-bearing mice. Values are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. \*\*\* Significantly different compared with the EAC control group at *p* < 0.001.

2.4.3. Effect of the Investigated Ceramides on the Serum Levels of Vascular Endothelial Growth Factor B (VEGF-B) and Tumor Necrosis Factor (TNF-α) and the Expression of Midkine (MDK) in the Tumor Tissue

The serum levels of vascular endothelial growth factor B (VEGF-B) and the tumor necrosis factor (TNF-α) were assessed by ELISA (Figure 4). Both markers were significantly increased in the EAC control group compared with the normal group (*p* < 0.001). The levels of VEGF-B were significantly decreased upon treatment by ceramide **A** (**1**) (1 and 2 mg/kg) and ceramide **B** (**2**) (1 mg/kg) (*p* < 0.01). The most significant decrease in the levels of VEGF-B in comparison with the EAC control group was recorded in the group treated by 2 mg/kg of ceramide **B** (**2**) (*p* < 0.001) (Figure 4A). Similarly, the levels of TNF-α showed a significant decrease in all treated groups: *p* < 0.05 in groups 3, 4, and 5 (1 and 2 mg/kg of ceramide **A** (**1**) and 1 mg/kg of ceramide **B** (**2**)) and *p* < 0.01 in group 6 (2 mg/kg of ceramide **B** (**2**)) (Figure 4B).

The levels of expression of midkine (MDK) in the tumor tissue were determined by real-time PCR. MDK was significantly upregulated in the EAC control group compared with the normal level (*p* < 0.001). The expression levels were significantly decreased in the groups treated with both doses of ceramide **A** (**1**) (1 and 2 mg/kg) and the lower dose of ceramide **B** (**2**) (1 mg/kg) (*p* < 0.01). The group treated with the higher dose of ceramide **B** (**2**) (2 mg/kg) showed the most significant downregulation of MDK compared with the EAC control group (*p* < 0.001) (Figure 5).

**Figure 4.** Effect of treatment with ceramide **A** and ceramide **B** at two dose levels (1 mg/kg and 2 mg/kg I.P) on the serum levels of (**A**) vascular endothelial growth factor B (VEGF-B) and (**B**) tumor necrosis factor-α (TNF-α). Values are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. # Significantly different compared with the normal group at *p* < 0.05. ## Significantly different compared with the normal group at *p* < 0.01. ### Significantly different compared with the normal group at *p* < 0.001. \* Significantly different compared with the EAC control group at *p* < 0.05. \*\* Significantly different compared with the EAC control group at *p*< 0.01. \*\*\* Significantly different compared with the EAC control group at *p* < 0.001.

**Figure 5.** Effect of treatment with ceramide **A** and ceramide **B** at two dose levels (1 mg/kg and 2 mg/kg I.P) on the expression of midkine (MDK) in the tumor tissue. Values are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. # Significantly different compared with the normal group at *p* < 0.05. ## Significantly different compared with the normal group at *p* < 0.01. ### Significantly different compared with the normal group at *p* < 0.001. \*\* Significantly different compared with the EAC control group at *p* < 0.01. \*\*\* Significantly different compared with the EAC control group at *p* < 0.001.

The VEGF members are key promotors of angiogenesis and lymphangiogenesis in malignancies. The level of VEGF-B in plasma was reported as a sensitive marker in breast cancer [38]. Overexpression of VEGF-B was found to promote metastasis in patients with pulmonary [39] and bladder [40] cancers. A higher expression of VEGF-B was also correlated with multiple tumors and positive vascular invasion in hepatocellular carcinoma patients [41]. Zhu et al. [42] suggested that downregulation of VEGF-B signaling may enhance the antitumor effect of resveratrol against pancreatic cancer. VEGF-B acts through binding to vascular endothelial growth factor receptor-1 (VEGFR1), leading to downstream activation of the angiogenetic and proliferative pathways including P38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK)/MAPK, protein kinase B/serine threonine protein kinase (PKB/AKT), and phosphoinositide 3-kinase (PI3K) [43,44]. On the other hand, the role of TNF-α in cancer has been extensively investigated. It is known to be a double player that has a marked effect in tumor progression on one hand but also may act as a pro-apoptotic agen<sup>t</sup> through activation of the c-Jun N-terminal kinase (JNK) pathway [45,46]. TNF-α is a major pro-inflammatory cytokine secreted by tumor-associated macrophages (TAMs) and by breast cancer cells. It is involved in all stages of the development of breast cancer, including tumor cell proliferation, epithelial-to-mesenchymal transition, metastasis, and recurrence [47]. Higher serum levels of TNF-α were reported in breast cancer patients compared with healthy individuals [48]. Additionally, the levels of TNF-α showed a correlation with the tumor stage in breast cancer patients [49–51]. TNF-α also activates nuclear factor kappa B (NFkβ) and induces Jagged1 expression, leading to activation of Notch signaling [52]. In a recent study conducted on a mammary carcinoma model, downregulation of TNF-α was associated with reduced VEGF, interleukin 6 (IL-6), interferon *γ* (IFN-*γ*), Jagged1, and shutting the Notch signaling pathway associated with enhanced apoptosis and declined angiogenesis [53].

MDK is a heparin-binding growth factor that is abnormally expressed in many human cancers, and it was found to mediate several tumor physiological processes involving cell growth, metastasis, migration, and angiogenesis. MDK is considered a key player in cancer progression and is proposed as a potential therapeutic target [54]. MDK expression

is induced by cytokines and growth factors, mainly by TNF-α [55], and it acts through activating the NFkβ and MAPK/PI3K proliferative pathway [54]. In clinical studies, MDK was considered a potential prognostic biomarker in solid tumors [56]. In breast cancer patients, it was suggested as both a diagnostic and a prognostic biomarker [57,58]. Interestingly, MDK is a potent proangiogenic factor that promotes tumor angiogenesis [59] and is thought to play a role in controlling the plasma bioavailability of VEGF-A [60]. MDK was also suggested to be implicated in the hypoxia-induced tumor angiogenesis [54,61]. The metastatic effects of MDK are most probably mediated by its combined proinflammatory, angiogenic, and mitogenic functions [62–64].

It is noteworthy that the group treated with the higher dose (2 mg/kg) of phytosphingosine ceramide **B** (**2**) expressed the most pronounced decrease in all the biochemically determined markers relative to the EAC control group, as mentioned above. The serum levels of VEGF-B and TNF-<sup>α</sup>, as well as the expression levels of MDK in the mice treated with 2 mg/kg of ceramide **B** (**2**), revealed the least significant difference compared with the normal (negative control) group (*p* < 0.05). This agrees with the findings of Kwon et al. [65], who reported that a phytosphingosine derivative exhibited an anti-angiogenic effect through markedly decreasing VEGF-induced proteolytic enzyme production, VEGFinduced chemotactic migration, and capillary-like tube formation.

2.4.4. Effect of the Investigated Ceramides on the Expression of the Apoptotic Markers p35, Bax, and Caspase 3 as Determined by Immunohistochemistry in the Tumor Tissue

Treatment with ceramide **A** (**1**) and **B** (**2**) at both doses augmented the expression of p53, with a significant difference from the control EAC group (*p* < 0.001) (Figure 6). Similarly, the expression of Bax was raised after treatment with both ceramides at the given doses (1 at 2 mg/kg), with a significant difference compared with the control EAC group (*p* < 0.001). The highest expression was observed in the group that received ceramide **B** (**2**) (2 mg/kg) (Figure 7).

(**A**)

**Figure 6.** *Cont*.

135

**Control Ceramide A (1 mg/kg) Ceramide A (2 mg/kg)** 

**Figure 6.** Effect of treatment with ceramide **A** and ceramide **B** at two dose levels (1 mg/kg and 2 mg/kg I.P) on the expression of p53. (**A**) Representative photomicrographs of p53 assessed immunohistochemically on day 21 in EAC-bearing female mice (40× magnification). (**B**) Optical density of positive immunohistochemical reactions (brown), determined using ImageJ. Values are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. \*\*\* Significantly different compared with the EAC control group at *p* < 0.001.

**Ceramide B (1 mg/kg) Ceramide B (2 mg/kg)** 

**Figure 7.** *Cont*.

(**B**)

**Figure 7.** Effect of treatment with ceramide **A** and ceramide **B** at two dose levels (1 mg/kg and 2 mg/kg I.P) on the expression of p53. (**A**) Representative photomicrographs of Bax, assessed immunohistochemically on day 21 in EAC-bearing female mice (40× magnification). (**B**) Optical density of positive immunohistochemical reactions (brown) determined using ImageJ. Values are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. \*\*\* Significantly different compared with the EAC control group at *p* < 0.001.

The relationship between ceramide and p53 is very complex, and the mechanisms underlying their coregulation are diverse and not fully characterized [66]. The p53 protein was established as a tumor suppressor [67]. It has been assumed that p53 exerts its effect by inducing apoptosis [68]. Cancer research was concerned with both the p53 and ceramide pathways in the regulation of cell growth, cell cycle arrest, and apoptosis [69]. Therefore, the present study investigated the connection between exogenous ceramide uptake and the levels of p53.

An association between p53 and ceramide was observed upon investigation of the cellular response to folate stress. Stressing the A549 cells caused p53-dependent activation of de novo ceramide biosynthesis and C16-ceramide elevation followed by apoptosis [70]. Coadministration of C6-ceramide with vincristine caused apoptosis in multiple cell lines. Significant activation of p53 was detected in these cells, leading to apoptosis [71]. C2- ceramide was shown to induce cell death via elevation of p53, a subsequent increase in the Bax/Bcl-2 ratio, and caspase activation [72].

Likewise, the expression of caspase 3 showed significant elevation compared with the control EAC (*p* < 0.001) in all treatment groups (Figure 8). This finding is in agreemen<sup>t</sup> with a previous study which reported that phytosphingosine can potently induce apoptotic cell death in human cancer cells via activation of caspase 3, 8, and 9, mitochondrial translocation of Bax, and the subsequent release of cytochrome c into the cytoplasm, providing a potential mechanism for the anticancer activity of phytosphingosine [73]. Additionally, sphingosine was reported to mediate apoptosis in various cancer cell lines through a caspase-dependent mechanism as well as truncation of Bax, which promotes pro-death activity [74].

**Figure 8.** Effect of treatment with ceramide **A** and ceramide **B** at two dose levels (1 mg/kg and 2 mg/kg I.P) on the expression of caspase 3. (**A**) Representative photomicrographs of caspase 3, assessed immunohistochemically on day 21 in EAC-bearing female mice (40× magnification). (**B**) Optical density of positive immunohistochemical reactions (brown) determined using ImageJ. Values are expressed as mean ± SD. All data were analyzed using ANOVA followed by a Bonferroni post hoc test. \*\* Significantly different compared with the EAC control group at *p* < 0.01. \*\*\* Significantly different compared with the EAC control group at *p* < 0.001.

Ceramide is involved in the induction of apoptosis and growth arrest in breast cancer [75]. The mechanism of ceramide-induced apoptosis involves elevated ceramide levels in the mitochondria, resulting in mitochondrial dysfunction, including a loss of cytochrome c [76]. Mitochondrial apoptosis is dependent on the increased mitochondrial outer membrane permeability. This permeability is enhanced by proteins such as Bax [77]. Therefore, channel formation by ceramide is an upstream event to the induction of apoptosis [78]. Just after the passage of ceramides into the mitochondria, many pro-apoptotic proteins are released into the cytoplasm, primarily cytochrome c [79]. Cytochrome c binds to Apaf-1 (apoptotic protease-activating factor-1). As a result, inactive procaspase-9 is cleaved into active caspase-9. Caspase-9 stimulates caspase-3, which is the crucial step for the caspase cascade in intrinsic apoptosis [80]. The start of intrinsic apoptosis is associated with a rise in mitochondrial ceramide levels [81]. Moreover, exogenous ceramide addition to cells induces apoptosis and DNA fragmentation [82]. Ceramide has been considered a crucial performer in the extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is activated by the death receptors through interaction with their ligands or by inducing receptor clusterization [83]. Acid sphingomyelinase catalyzes the hydrolysis of sphingomyelin into ceramide, consequently generating ceramide-rich stages and the subsequent clusterization of death receptors that enables the formation of a death-inducing signaling complex and caspase activation [84].
