Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model

Persistent infection with high‐risk human papillomavirus (HR‐HPV) is a well‐established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR‐HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17β‐estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO‐saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR‐218 and the oncomiRNA miR‐21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non‐transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.


| INTRODUCTION
HPV E7 oncoprotein plays a pivotal role in cervical carcinogenesis since their continuous expression is necessary for the initiation and maintenance of the malignant phenotype. 1The E7 oncoprotein is an efficient cell cycle deregulator by targeting for degradation, among other proteins, Rb protein family members which leads to the E2F transcription factor release and S-phase progression. 2Additional factors such as chronic hormonal contraceptives, multiparity and nutritional deficiency, are necessary to the progression of Cervical Cancer (CC).Notably, the K14E7HPV16 (K14E7) mouse model has emerged as a valuable tool in elucidating the mechanisms underlying cervical carcinogenesis. 3,4Previously, we have observed that a single 17βestradiol (E 2 ) dose within intraperitoneal cavity is sufficient to develop cervical severe hyperplasia and mild dysplasia in 1.5month-old K14E7 mice (manuscript in preparation).Interestingly, in this work we found that 3-month-old K14E7 mice develop CIN1 after a single hormonal treatment performed at 2.5 months of age.
MicroRNAs (miRNAs) are small noncoding molecules intricately involved in the mechanism of negative gene expression regulation.They are increasingly acknowledged as potential biomarkers for diagnosing and prognosticating various diseases, including CC. 5 MiR-21, an oncomiRNA, is overexpressed in CC; however, its expression has been found to be decreased in early cervical lesions. 6On the other hand, miR-218 (a known tumor suppressor) has been described to be negatively regulated in CC; indeed, the level of expression of this miRNA changes with the degree of cervical lesion. 5,6tients diagnosed with CIN 1 or 2, usually are typically monitored cytologically or colposcopically and then are subsequently treated only if the lesion persists or progresses after 18 to 24 months.In the case of persistent lesions, the medical procedures used are highly invasive (either using laser ablation or cryotherapy).
New treatment alternatives to mitigate unwanted side effects include natural products.Among these, algae obtained from marine environments stand out as a prolific source of active compounds.Within red algae, the genus Laurencia is one of the richest sources of novel compounds among red algae. 7urencia johnstonii Setchell & Gardner (1924) (L.johnstonii) is a seaweed endemic to the Gulf of California, Mexico, and it has been shown that have cytotoxic and antitumor properties. 7,8García-Davis et al. 7 used three-dimensional (3D) cultures of human breast cancer explants to evaluate the antitumoral effect of an ethanolic extract of L. johnstonii (ELj) and the sesquiterpene lauritenol.After studied the metabolic and histopathological effects of this compounds in human breast cancer explants and MCF-7 cell line, this group observed a dose-dependent inhibition of the metabolic activity and increase of apoptosis.
The aim of this work was to evaluate the effect of an ethyl acetate crude ELj in early cervical lesions using the transgenic mice model expressing E7 viral oncogene.

| Female K14E7HPV (K14E7) transgenic and FvB/n (nontransgenic control) mice
Maintenance and genetic characterization of K14E7 transgenic mice have been previously reported. 3,4FvB/n mice were originally obtained from Jackson Laboratory.Animals were housed and bred in pathogen-free equipment at the animal facilities (UPEAL-CINVESTAV-IPN), according to the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International) regulations.

| Estrous cycle determination
Using exfoliative cytology technique, Estrous cycle was determined in 48 3-month-old female control and transgenic mice as described elsewhere. 4After Hematoxylin-Eosin staining, cytological preparations were analyzed by bright field microscopy.S1).The concentration of 17β-estradiol (833 ng/day) used for the hormonal treatment was previously described elsewhere. 3On the other hand, and before ELj treatment, additional FvB IND TX and K14E7 IND TX experimental groups were hormone treated in the same manner.Experimental groups FvB, K14E7, FvB TX, and K14E7 TX, were used as controls of hormone treatment.All mice were killed at 3-month-old.

| Treatment with the ELj
Twelve 2.5-month-old K14E7 virgin female mice and twelve FvB/n mice (experimental groups K14E7 TX and FvB TX), were treated with the ELj intraperitoneally (IP; 30 mg/kg weight).A dose of the extract previously diluted in an injectable DMSO-saline solution (0.1 mg of ELj in DMSO/ mouse per day) was used every third day, until completing seven doses and 3-month-old (Figure S1).For the FvB IND TX and K14E7 IND TX experimental groups, and after E 2 induction of 2.5-month-old mice in Diestrus and confirming that all the animals were in estrous, the ELj treatment were performed in the same manner.The dosage of ELj was calculated by extrapolating the results obtained previously in tumoral cell lines and previous studies reported in the literature on the use of Laurencia in animal models for different purposes, including antitumor effects. 7,9All mice were killed at 3-month-old.Animal without hormonal induction were killed in Estrous at 3-month-old.

| Cervical tissue procurement and histopathological analysis
All mice were euthanized in a carbon dioxide euthanasia chamber and subsequent cervical dislocation according to institutional and international regulations (AAALAC; UPEAL-CINVESTAV-IPN; NOM-062-ZOO-1999).After paraformaldehyde fixation, cervix of animals was dehydrated, and paraffin embedded.The entire female genital tract was cut in five microns thick sections, stained with hematoxylineosin and evaluated as described elsewhere. 4

| Histopathological analysis of murine liver, spleen, and kidney to rule out ELj toxicity
To confirm indirectly that the ELj did not induce secondary damage in the murine model, the histopathology analysis of the liver, spleen, and kidney of all experimental groups was performed.Following euthanasia, the extraction of these organs of six mice from each experimental group was carried out.Tissues were paraformaldehyde fixed, dehydrated, and paraffin embedded as described above for cervical tissues.Five-microns thick sections were performed, stained with hematoxylin-eosin and further evaluated by an expert pathologist adhering to established protocols described elsewhere. 4

| Immunohistochemistry
Histological sections were de-paraffinized, rehydrated, and analyzed using the Super Sensitive Polymer-HRP IHC Detection kit (BioGenex) according to manufacturer′s instructions.Sections were incubated for 12-16 h at 4°C with primary antibody against cell proliferation marker PCNA or apoptosis marker Cleaved Caspase 3 (Santa Cruz Biotechnology) at 1/50 dilution.Tissues without primary antibody were included as negative staining controls.

| Digital analysis of immunohistochemical assays
For semiquantitative analysis of immunohistochemical experiments, representative images were obtained using a DFC290 HD digital camera (Leica Microsystems) as described elsewhere. 4

| Total RNA isolation and miRNA quantification by real-time quantitative PCR (RTqPCR)
Total RNA was isolated from cervix obtained from three mice of each experimental group using the TRIzol method according to manufacturer's instructions (Thermo Fisher Scientific, Inc.).

| Quantification of miR-218 and miR-21 expression levels by RTqPCR
To detect the expression levels of mature miR-218 and miR-21, the TaqMan ® MicroRNA assays (miR-218 [ID 000521]; miR-21 [ID 000397]; Applied Biosystems) were used as described elsewhere. 5Expression levels were normalized to the endogenous control snoRNA202 (ID 001232; Applied Biosystems).The data was analyzed using the equation described by the 2 C -ΔΔ T method. 10

| Statistical analysis
Statistical analysis of miRNAs was conducted utilizing the Mann-Whitney U test.For the immunohistochemical assays, statistical analysis was performed based on relative quantification expressed as a percentage of positive signal for PCNA or Cleaved Caspase 3 proteins, followed by ANOVA-post hoc Bonferroni analysis.Statistical significance was defined as p ≤ 0.05.

| Determination of the families of metabolites present in the ELj
Following the TLC analysis of ELj and subsequent utilization of general revelators, numerous spots were discerned, indicating the presence of different metabolites.These include steroidal saponins and triterpenes, phenols, catechol-derived tannins, alkaloids, retinol, flavonols such as xanthones and flavones, aldehydes, and ketones.
Exhaustive phytochemical analyzes (structural elucidation, MPLC, HPLC, and LC-MS) have been conducted, and detailed findings will be presented in a forthcoming manuscript (manuscript in preparation).

| ELj treatment did not induce evident histological alterations in murine liver, spleen, and kidney
The histopathology of liver, spleen, and kidney sections from mice with or without ELj treatment was performed.In the case of liver obtained from ELj-treated mice, the normal integrity of the portal triad (hepatic artery, portal vein, and bile duct) was observed as well as the absence of infiltrate inflammatory and signs of necrosis.
Likewise, the loss of tissular architectural integrity was ruled out compared to the nontreated-FvB mice (control group; Figure S2).In spleen, it was observed the normal integrity of the capsule was evaluated as well as a well-defined differentiation of the white and red pulp.The presence of inflammatory infiltrate and signs of necrosis was also ruled out.All experimental groups showed normal splenic architecture, except for K14E7 mice in which the white and red pulp was observed not well differentiated, probably due to E7 oncoprotein expression.Interestingly, this histological alteration seemed to disappear in the ELj-treated K14E7 experimental groups (Figure S3).Finally, kidney obtained from all experimental groups exhibited intact renal capsule integrity, integrity of the renal capsule, absence of glomerular damage (homogeneity of the interglomerular space), normal integrity of the collecting tubules, as well as the absence of both, inflammatory infiltrate and necrosis (Figure S4).

| Treatment with the ELj restored the architecture of the E 2 -induced-K14E7 cervical epithelium and decreased the development of metaplasia
The histopathology of the exocervix and the transformation zone (TZ) of the cervical tissue of each of the experimental groups was examined.As expected, the exocervical tissue of the female mice of control group (FvB) showed a healthy cervical epithelium.Similarly, in FvB TX mice no obvious alteration was observed in the cervical stratified epithelium.(Figure 1A and Table 1).In comparison, the FvB (Figure 1B and Table 1).

| ELj treatment diminished the cell proliferation levels in exocervix of K14E7 IND TX mice
The cell proliferation levels were determined detecting the PCNA protein as a molecular marker (Figure 2A).As expected, the exocervix Additionally, a significant decrease in proliferation was observed between the K14E7 IND and the K14E7 IND TX groups (Figure 2B).

| ELj treatment restored the normal cell proliferation levels in squamous-columnar junction (TZ) of E 2 -induced K14E7 mice
In the TZ of FvB control mice, a proliferation signal was observed at the basal layer of the squamous epithelium, while in the columnar epithelium the signal was very little or null (Figure 3A).In the cervical epithelia of FvB IND mice, proliferating cells were evident in various layers, encompassing both squamous and columnar epithelia.Notably, FvB TX and FvB IND TX mice exhibited a decrease in cell proliferation levels, with positive nuclei predominantly confined to the basal and suprabasal layers, resembling the pattern observed in controls (Figure 3).In addition, we observed statistically differences F I G U R E 1 Histopathological analysis of cervical tissue obtained from the experimental groups.(A) Representative histopathology images of exocervix obtained from 3-month-old FvB/n (FvB) and K14E7 transgenic mice.Cervical tissue of ELj-treated and ELj-treated/E 2 induced K14E7 transgenic mice (K14E7 TX and K14E7 IND TX, respectively), compared to control group, displayed mild hyperplasia, and enlarged nuclei restricted to the upper layers; interestingly, no koilocyte-like cells were observed.On the other hand, the K14E7 transgenic mice (K14E7 and K14E7 IND) showed abundant inflammatory infiltrate (ii; solid arrows) and koilocyte-like cells (k; empty arrows).(B) Representative histopathology images of the transformation zone (TZ) of the cervix obtained from 3-month-old FvB and K14E7 transgenic mice.K14E7 transgenic mice showed severe metaplasia and moderate dysplasia while the K14E7 IND mice showed poorly defined TZ, severe metaplasia with mitotic figures, mild dysplasia, loss of differentiation, and the presence of koilocyte-like cells (empty arrows).K14E7 TX showed a poorly defined TZ and moderate metaplasia.K14E7 IND TX mice display moderately defined TZ and mild metaplasia.These results are representative of three independent experiments (please see Table 1 3B).These K14E7 groups showed cells with a positive nucleus, randomly distributed in almost all layers of the TZ (Figure 3A); this being more evident in the K14E7 IND mice.In the case of the K14E7 TX and K14E7 IND TX groups, a decrease in cell proliferation levels, restricted to the basal and suprabasal layers of the epithelium, can be observed.The digital quantification showed a statistically significant decrease in the K14E7 IND TX group mice (Figure 3).IND TX groups treated with ELj, recovered their levels of apoptosis as compared to controls (Figure 4A).In Figure 4B, it can be seen a significant decrease in the levels of Cleaved Caspase 3 in the K14E7 IND group compared to the control group FvB; however, these levels increased after the administration of the ELj extract.In fact, these mice showed Cleaved Caspase 3 expressing levels even higher than the FvB control group.In the TZ, similar results to those obtained in the exocervix can be observed.There is a significant decrease in Cleaved Caspase 3 levels in the K14E7 IND group compared to the FvB control group; however, after administration of ELj extract in the K14E7 IND TX group, a significant increase in apoptotic cells was observed, with protein levels being higher than the control FvB group (Figure 5B).comparable to FvB controls (Figure 6).

| DISCUSSION
Early diagnosis and treatment of cancer, in most cases, represents a high probability of successfully eradicating this disease. 1,11Therefore, it is necessary the search for new, noninvasive treatments for early cervical lesions, complementary to the existing ones, such as the use of natural products including the seaweed L. johnstonii Setchell & Gardner. 7,8,12 the present work, young female mice of 3 months of age Mild to moderate hyperplasia; few mitotic figures; koilocytes not seen; mild or absent dysplasia as flattened nuclei (na) are observed, suggesting differentiation in the upper layers 100 alkaloids, retinol, and flavonols, some of which it has been demonstrated to possess antitumor properties. 9,13Before this, we evaluated the histopathology of liver, spleen, and kidney of ELjtreated mice to verify any secondary damage, determining that there were no histopathological alterations (Figures 2-4).
The main goal of this work was to analyze the histopathological changes in the cervical tissue of the E 2 -treated K14E7 (K14E7 IND) murine model with ELj treatment.In K14E7 transgenic mice (K14E7 and K14E7 IND), the presence of an inflammatory infiltrate (a recognized risk factor for the development of cancer) was observed.
Additionally, K14E7 IND mice developed moderate hyperplasia and mild dysplasia, suggesting a CIN 1-like lesion.Interestingly, ELjtreated mice showed that the cervical epithelium practically recovered its normal architecture, decreasing the hyperplasia and dysplasia, as well as inflammatory infiltrate observed in FvB IND and K14E7 IND, suggesting that ELj probably is inhibiting both, the action of E 2 and the E7 oncoprotein.The E7 oncoprotein induces a less differentiated transformation zone (TZ), an effect that is increased by the presence of E 2 inducing metaplasia, condition that was diminished in the ELj-treated groups, where the TZ was observed well defined.
It is well known that E7 oncoprotein deregulates the cell cycle and amplifies the proliferative stimulus propagated by estrogen, which by itself is a recognized mitogen. 3,14The histopathological changes observed in both the exocervix and the TZ of the K14E7 IND mice could be due to increased cell proliferation levels and increased genomic instability (Figure 2), which in turn alter the normal architecture of the epithelium, causing premalignant lesions. 15 inhibiting cell proliferation and increasing apoptosis levels in vitro. 9,12,16[19][20] According to our observations, a single E 2 stimulus was enough to decrease the levels of apoptosis in both the FvB and K14E7 mice.Previous studies have demonstrated that E 2 has the ability to inhibit apoptosis mediated by estrogen receptors. 19,21terestingly, a significant increase in the levels of apoptosis was observed in the ELj-treated K14E7 mice.Different studies have confirmed that several species of Laurencia, such as Laurencia viridis and Laurencia papillosa, are capable of inducing apoptosis in cell lines derived from breast cancer, an effect that is attributed to the sesquiterpenes and diterpenes that are also present in L.
johnstonii. 8,12,16,22,237][28] It should be noted that, to date, the accurate mechanism by which the E7 oncoprotein regulates the expression of this miRNA has not been reported.
K14E7 TX mice showed that the expression of miR-218 tends to increase with respect to that observed in untreated K14E7 mice, reaching expression levels similar to controls, probably due to terpenoids and flavonoids metabolites of the ELj extract, which had shown the ability to increase the expression of this miRNA in many types of cancer studies. 24,25single E 2 dose in FvB induced a significant increase of miR-21; this coincides with previous reports. 29In K14E7 IND mice the expression of miR-21 was found to be overexpressed, contrary to Pedroza-Torres et al. 30 and Liu et al. 31 where it is observed that in low-grade cervical lesions this miRNA is underexpressed; however, in these studies the estrogenic action was not considered, and patients were infected with different HPV genotypes.According to our results, it has been reported that the E7 oncoprotein induces the overexpression of miR-21, an effect that is enhanced by estradiol. 29 observed a decrease in the expression of this oncomiRNA in FvB

2. 2 |
Obtaining the extract of L. johnstonii Setchell & Gardner (ELj) L. johnstonii Setchell & Gardner (1924) specimens were collected during the months of March through June at the localities of "Agua de Yepiz" (Lat 24°17′ 20″ N, Long 110°37′ 56″ West) and "El Sauzoso" (Lat 24°18′ 55″ N, Long 110°38′ 28″ W) at "La Paz" bay (2021-2022).The specimens were collected from the subtidal zone by free diving and only epiphyte-free algae were meticulously selected.The material was transported to the Interdisciplinary Center for Marine Sciences of the National Polytechnic Institute (CICIMAR-IPN).The algae were dried in the shade, crushed, weighed, and stored at −20°C until transported to the facilities of the National School of Medicine and Homeopathy of the National Polytechnic Institute (ENMyH-IPN).Taxonomic identification of the specimens was conducted based on specific features at the UABCS Marine Botany Laboratory. 8A representative specimen taken from the recollection, was cataloged, and deposited in the Phycological Herbarium of the Autonomous University of Baja California Sur (UABCS) with the catalog number FBCS 20252.To obtain ELj, we utilized a medium polarity solvent ethyl acetate which allowed us to obtain a greater variety of metabolites (polar to nonpolar nature).Thin layer chromatography (TLC) was used for the visualization and qualitative analysis of metabolites present in the ELj.For the identification of the families of metabolites present in the sample, Ceric sulfate, Liebermann-Burchard, Rosenthaler, Salkowski, Ferric hydroxide, Folin-Ciocalteu, Baljet, Ferric chloride, Dragendorf, Molisch, Carr-Price, Ammonium Hydroxide, 10% Sodium Hydroxide, and 2,4-dinitrophenylhydrazine (DNPH) were employed.
The experimental groups used in this study comprised six animals each, categorized as follows: (a) FvB/n mice in Estrous (FvB); (b) FvB/n mice induced (Diestrus mice induced with E 2 to Estrous) (FvB IND); (c) ELj-treated FvB/n mice in Estrous (FvB TX); (d) FvB/n mice induced and ELj-treated (FvB IND TX); (e) K14E7 transgenic mice in Estrous (K14E7); (f) K14E7 mice induced (K14E7 IND); (g) ELj-treated K14E7 mice in Estrous (K14E7 TX); (h) K14E7 mice induced and ELj-treated (K14E7 IND TX).Three animals from each experimental group were used for RNA purification and other three were used for tissue procurement.The animals in each group showed similar phenotypic and histological characteristics.
mice induced with E 2 (FvB IND) showed moderate hyperplasia, abundance of atypical nuclei with relaxed chromatin suggesting increased cell activity in upper layers.However, FvB IND TX mice presented cervical epithelium with mild hyperplasia together with a decrease in the number of atypical nuclei.Differentiated cells with flattened nuclei in upper layers similar to those of healthy cervical epithelium were also observed.Despite the presence of inflammatory infiltrate in both experimental groups, there was a discernible tendency for the cervical epithelium to revert to its natural structure following ELj treatment.On the other hand, K14E7 IND mice presented moderate hyperplasia, koilocytes, inflammatory infiltrate, lack of differentiation and CIN 1.In K14E7 TX mice, mild to moderate hyperplasia, possible CIN 1, sparse presence of koilocytes and no obvious inflammatory infiltrate was seen.Finally, and outstanding, the K14E7 IND TX mice showed mild to moderate hyperplasia, scanty mitotic figures, and the absence of koilocytes.In addition, these mice showed mild or absent dysplasia and flattened nuclei, suggesting differentiation in the upper layers.The TZ of FvB control mice contained a well differentiated stratified squamous epitheliumcolumnar epithelium junction (Figure 1B).In the FvB IND experimental group, the TZ exhibited poor definition due to mild metaplasia.Interestingly, both, FvB and FVB IND mice treated with the ELj, presented a well-defined TZ.Conversely, in K14E7 transgenic mice, it was possible to observe a poorly defined TZ due to a metaplasia process, this being more evident in the K14E7 IND group.The cervix of K14E7 IND TX mice showed a normal-like cervical epithelium of control group mice (FvB) showed cells in proliferation restricted to the basal layer of the epithelia.On the other hand, FvB IND mice showed cells in proliferation from basal to superficial layers in a nonhomogeneous way.While FvB TX mice showed no discernible decrease in cell proliferation, FvB IND TX mice exhibited a notable reduction in cell proliferation levels (Figure2A).In the four K14E7 groups, cells with a positive signal were observed in almost all layers of the epithelium, this being even more evident in the K14E7 IND group.K14E7 TX mice showed a decrease in proliferation, similar to that observed in control groups; on the other hand, in K14E7 IND TX mice we can detected a decrease in proliferation compared to K14E7 IND mice, this proliferation was restricted to the basal and suprabasal layers of the cervical epithelium (Figure2A).A significant difference was found in the proliferation levels of the E 2 -treated groups (FvB IND, K14E7 IND), as well as in the FvB IND TX and K14E7 groups.

3. 6 |
ELj treatment restores apoptosis levels in cervical tissue (exocervix and TZ) of E 2 -induced K14E7 mice comparable with FvB control mice As expected, E 2 -treated FvB and K14E7 experimental groups (FvB IND and K14E7 IND mice) showed diminished apoptotic levels in comparison with their respective controls (FvB and K14E7); in addition, these groups did not show important changes in apoptosis levels after ELj treatment.Interestingly, the FvB IND TX and K14E7

3. 7 |
Effect of ELj treatment on the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21 molecular biomarkers FvB IND mice showed a significant increase in both, miR-218 and miR-21 expression levels in comparison with FvB controls; subsequently, the administration of ELj treatment significantly reversed these increases, reaching relative expression levels close to controls (Figure 6).In contrast, after E 2 -treatment K14E7 IND group showed decreased levels of miR-218 and increased expression of miR-21.Interestingly, those E 2 -induced groups and treated with ELj (FvB IND TX and K14E7 IND TX) showed a tendency to recover their relative expression levels of both, miR-218 and miR-21 microRNAs,

(equivalent to 20 -
25 years in humans) expressing the HPV16 E7 oncoprotein and exposed to hormonal stress were used; these mice present CIN 1 lesions.These mice are excellent model to in vivo studies related to therapy of early cervical malignant lesions hence we evaluate the therapeutic potential of a crude extract of L. johnstonii Setchell & Gardner (ELj).The ELj contains a variety of metabolites including steroidal and triterpene saponins, phenols, T A B L E 1 Histological analysis of cervical tissue in ELj-treated mice.hyperplasia; abundance of atypical nuclei with relaxed chromatin suggesting cellular activity in upper layers; presence of inflammatory infiltrate 100 FvB TX (n = 3) Normal stratified epithelia 100 FVB IND TX (n = 3) Mild cervical atrophy; decrease in the number of atypical nuclei; differentiated cells with flattened nuclei (na) are seen in upper layers similar to those seen in healthy cervical epithelium.The inflammatory infiltrate (ii) is preserved but at a moderate level.100 K14E7 (n = 6) Moderate hyperplasia; possible CIN 1; inflammatory infiltrate; presence of koilocytes 100 K14E7 IND (n = 3) Moderate hyperplasia; mild mitotic figures; CIN 1; inflammatory infiltrate; the presence of koilocytes is increased 100 K14E7 TXK14 E7 TX (n = 3) Mild to moderate hyperplasia; mild dysplasia, possible CIN 1; sparse presence of koilocytes.No obvious inflammatory infiltrate is seen.100 K14E7 IND TX (n = 3) This suggests that the ELj conduces to recover the normal cervical epithelium by reducing the rate of cell proliferation.This decrease coincides with previous studies where it has been shown that the terpenoids present in different Laurencia species, are capable of F I G U R E 2 Regression of the cell proliferation levels in exocervix of K14E7 IND TX mice after ELj treatment.(A) Immunohistochemical detection of the PCNA protein in cervical tissue of 3-month-old mice.PCNA protein was detected mainly in the nuclei of cervical tissue basal cells of FvB mice while in K14E7 IND or K14E7 TX mice showed positive signal in basal and suprabasal cells (empty arrows); in addition, K14E7 IND TX showed positive proliferative cells in basal epithelial layer.(B) Relative amount of PCNA positive signal.These results are representative of three independent experiments.Statistically significant: * (p ≤ 0.0001).n = 3 mice were used per experimental group.BM, basal membrane.

. 24 F
observed that a sesquiterpene-treated mouse model with melanoma overexpressed Bak, Caspase-9 and p53 proteins.Additionally, it has been demonstrated that flavonols, molecules found also in ELj, are able to increase the expression and subsequent activation of Caspase 3I G U R E 3 Regression of cell proliferation levels in the transformation zone of the cervical tissue obtained from K14E7 IND TX mice after ELj treatment.(A) Immunohistochemical detection of the PCNA protein in the transformation zone (TZ) of the cervix of 3-month-old mice.PCNA protein was detected mainly in the nuclei of cervical tissue basal cells of FvB mice while in K14E7 IND or K14E7 TX mice showed positive signal in basal and suprabasal cells (empty arrows); in addition, K14E7 IND TX showed positive proliferative cells in basal epithelial layer like control FvB mice.(B) Relative amount of PCNA positive signal.These results are representative of three independent experiments.Statistically significant: * (p ≤ 0.01).n = 3 mice were used per experimental group.BM, basal membrane.The microRNAs regulate at least one-third of all human genes, so it is easy to understand that the pathogenic properties of cancer cells, such as survival, uncontrolled cell proliferation, invasion, and metastasis, are regulated by them.There is experimental evidence that the secondary metabolites of natural extracts can regulate the expression of various miRNAs,25 making them excellent molecular biomarkers for assessing the biological activity of an extract.Given this context, we determined the expression levels of two molecular biomarkers of cervical cancer miR-218 and miR-21.The estrogen stimulus in the FvB IND mice induced a significant increase in the expression levels of the tumor suppressor miR-218.These results are probably due to a response from the young mice to compensate cell and tissue damage caused by hyperproliferation and decrease in apoptosis.Interestingly, FvB TX mice exhibited a trend toward regulating/maintaining miR-218 expression levels similar to those of the control group without estrogen, suggesting a potential inhibitory effect of ELj on estrogen-induced actions.

F I G U R E 4
The extract of Laurencia johnstonii restores the apoptotic levels in exocervix of K14E7 IND TX transgenic mice.(A) Apoptotic levels determined by Cleaved Caspase 3 were diminished in K14E7 IND TX mice similar to FvB control mice.Empty arrows: Apoptotic cells.(B) Digital analysis of apoptotic levels (relative Cleaved Caspase 3 labeling index) in murine exocervix.These results are representative of three independent experiments.Statistically significant: * (p ≤ 0.0001).n = 3 mice were used per experimental group.BM, basal membrane.
TX and K14E7 IND TX mice, this being statistically significant in FvB IND TX mice as compared to the FvB IND mice.This finding corroborates several studies demonstrating that alkaloids and phenolic compounds, detected in the ELj extract, can downregulate the expression of miR-21 in cancer cells.[32][33][34][35][36]Despite statistically significant differences in the expression of miR-218 and miR-21 results were mostly observed in the nontransgenic induced (FvB IND) mice versus nontransgenic induced treated mice (FvB IND Tx), our results corroborate that the metabolites present on the ELj extract do have the ability to modify the expression of these miRNAs.Additionally, it is well known that the sole action of the E7 oncoprotein can inhibit the expression of this and others tumor suppressors and enhance the expression of oncomiRNAs such as miR-21, that is why we do not rule out the possibility that E7 could also be interfering with the ELj mechanism of action.Given this, not only different doses of the ELj extract must be F I G U R E 5 The extract of Laurencia johnstonii restores the apoptotic levels in the Transformation Zone (TZ) of K14E7 IND TX transgenic mice.(A) Apoptotic levels determined by Cleaved Caspase 3 were diminished in K14E7 IND TX mice similar to FvB control mice.Empty arrows: Apoptotic cells.(B) Digital analysis of apoptotic levels (Cleaved Caspase 3 labeling index) in murine exocervix.These results are representative of three independent experiments.Statistically significant: * (p ≤ 0.0005).n = 3 mice were used per experimental group.BM, basal membrane.