PCR analysis of the effect of photodynamic therapy on breast tumors

Photodynamic therapy (PDT) is a promising therapeutic modality for treating cancer, including breast tumors. The oxidative damage caused by PDT culminates in cell death, induction of immune response, and the resulting destruction of the tumor. This study aimed to evaluate the gene expression profiling of genes BCL-2, BAX, and HER-2 and their proteins after PDT, associating it with the necrosis caused by this therapy under different fluences. Twenty-eight female rats received a single dose of 7,12-dimethylbenz (a) anthracene (DMBA - 80mg/kg), by gavage, for breast tumor induction. After the tumors grew, the animals were divided into four groups: G1 - control group – untreated breast tumor – and G2, G3, and G4 groups treated with PDT using Photogem@ as photosensitizer and interstitial irradiation, with fluences of 50J/cm, 100J/cm, and 150J/cm, respectively. Samples of tumors were harvested for histological examination by RT-qPCR. The RT-qPCR showed that the gene expression profiling of BCL-2, BAX, and HER-2 was not altered after PDT. Hemorrhagic necrosis and qualitatively greater vascular and cellular damage were observed and correlated positively with the fluence. PDT does not seem to induce the modulation of genes related to apoptosis. The results indicate that the type of cell death stimulated by PDT in breast tumor is necrosis.


Introduction
Photodynamic therapy (PDT) is a promising therapeutic modality used for the treatment of cancer and other ailments.
The induction of oxidative stress and formation of reactive oxygen species (ROS) result in organelle damage, culminating in cell death and destruction of the tumor or abnormal tissue after PDT (Oleinick & Evans, 1998). Apoptosis is a common mechanism of cell death in this case; however, the triggering events responsible for the PDT-mediated apoptotic response are not clear.
Understanding the mechanisms involved in PDT-induced apoptosis may improve therapeutic efficacy (Srivastava et al. 2001), contributing to the knowledge on treatment failures (Itoh et al. 2000;Senderowicz, 2004).
PDT could be used as an alternative treatment for the control of tumor growth or as an adjunct therapy to surgery, in order to avoid recurrence. A previous study conducted by our research group showed that PDT decreased the rate of tumor growth, although the remission of the tumor was not complete. Endogenous factors could be associated with the unsatisfactory results (Ferreira et al. 2012). As a consequence, the authors emphasized the need for more comprehensive studies, investigating the action of endogenous components in the success of PDT.
Some studies have shown that the anti-apoptotic protein Bcl-2 is the target of photosensitizers. Bcl-2 sequesters proapoptotic molecules, such as Bax and Bak, inhibit apoptosis. Moreover, Bcl-2 prevents the release of potent mitochondrial activators of the cytosolic death effector proteases, which mediate the intracellular proteolysis typical of apoptosis. Therefore, the downregulation of Bcl-2 and related proteins may result in triggering apoptosis (Srivastava et al. 2001;Kessel & Arroyo, 2007).
The discovery of the role of the HER2 gene was one of the milestones in breast cancer research over the past two decades. Breast cancer in women is one of the major causes of death in both developed and developing countries (Ahmed et al. 2012). It is estimated that the incidence of breast cancer has increased by 20% since 2008, and mortality by 14%, these data show that the spread of breast cancer is significant (Alteri et al. 2013;Pitta et al. 2021). HER2 + tumors exhibit faster growth and present higher metastatic capacity compared to HER2-tumors (Hicks et al. 2008). In addition, some authors have claimed that this gene seems to be an important contributor to the antiproliferative response to PDT, interfering with the necrosis and apoptosis processes and tumor destruction (Martinez-Carpio & Trelles, 2010).
The aim of this study was to investigate the gene profiling expression of BCL-2/Bcl-2, BAX/Bax, and HER2/erbB2 genes in tumors treated with PDT under different fluences compared to untreated tumors. The signals that trigger cell death and the cascade of events leading to apoptosis are today important targets in the search for new anticancer therapy.

Materials and Methods
This research is in accordance with the Ethical Principles in Animal Experimentation and was approved by the Ethics Committee of the Faculty of Veterinary Medicine and Animal Science-UNESP-Botucatu (protocol: 72/2009).

Experimental design
A total of 28 young Sprague-Dawley female rats weighing between 200-250 g was used in this study. The animals remained in the ideal conditions of temperature (22 ± 2°C), humidity (50± 20%), and light (12h light: 12h dark cycle). Feed and water were provided ad libitum. Breast cancer was induced by a single dose of 80 mg/kg 7,12-dimethylbenz (a) anthracene Haematoporphyrin (Photogem®; Photogem LLC Company, Moscow, Russia) was used as photosensitizer at 12 mg/kg, administered intraperitoneally to all the animals. Regarding PDT application, the light source was a laser diode Ceralas Ceramoptec 630®, Germany, at a wavelength of 630 nm. The amount used was 200 mW/cm2.
In order to perform the PDT, the animals were anesthetized intramuscularly with 20 mg xylazine 2% (0.2 ml/100 g bw; Vetbrands®, Brazil) and ketamine 50 mg (Vetaset, Fort Dodge, Brazil). The animals received the photosensitizer by intraperitoneal administration and 24 hours later, the laser irradiation was carried out using an optic fiber with a diffuser (1cm) coupled to a laser with 250 s (Group 2), 500 s (Group 3), and 750 s (Group 4) fluences. The diffusing fiber was inserted into the tumor central part, ensuring total tumor irradiation.
The animals were anesthetized with isoflurane 30 hours after irradiation and then euthanized by transection of the jugular veins.

Histopathology
A tumor fragment of each animal was collected for histopathological preparation. The histopathological material was fixed with 10% buffered formalin, then embedded in paraffin, and stained with hematoxylin and eosin (HE). In the histological preparations, morphological differences and points of necrosis in the treated tumor groups were analyzed and compared to the Research, Society and Development, v. 10, n. 12, e459101220468, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i12.20468 4 control group. A comparison was also performed between the fluences used and the depth of necrosis observed in the breast tissue.

Reverse transcription-quantitative PCR (RT-qPCR)
Samples of tumors were collected and immediately frozen in liquid nitrogen and then conditioned at -80 ○C. The extraction of total RNA from the samples was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer. Then, the concentration and purity of the RNA was assessed using a spectrophotometer (NanoVue -EG).
Complementary DNA was synthesized from 0.1 g of total RNA using the High-Capacity Kit (Applied Biosystems, Branchburg, New Jersey, USA), according to the instructions of the manufacturer. The reactions were incubated at 25 °C for 10 min and 37 °C for 120 min, and then remained at 4 °C. The samples were stored at -20 ○C.
Each tube contained 2 µL of cDNA template, 5 µL of TaqMan 2X Master Mix (Applied Biosystems), 2.5 µL of water, and 0.5 µL of 20X primers/probes (Assays-on-Demand gene expression products, Applied Biosystems). The reaction was performed under the following thermal cycling conditions: 94 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, and 60 °C for 1 min.
Fluorescence data were collected during each annealing/extension step. The efficiency of the primers was assessed with standard curves, with the β-actin, BCL-2, BAX, and HER-2 primers presenting efficiencies above 90%. The reactions were performed using the 7500 FAST Real-Time PCR System (Applied Biosystems) and the SDS software, version 1.2.3 (Sequence Detection Systems 1.2.3, 7500 Real-Time PCR Systems, Applied Biosystems). In order to prevent contamination, a negative control (without DNA) was added for each plate. The relative gene expression data were analyzed using the 2−∆∆CT method (Livak & Schmittgen, 2001). An RQ (Relative quantification) <0.5 was considered as a decreased gene expression, and an RQ> 2.00 was regarded as an increased gene expression.

Statistical analysis
Statistical analyses were performed using SAS software, version 9.2 (Statistical Analysis System, SAS Institute, Cary, NC, USA). The results were compared using the test of analysis of variance (ANOVA) to detect statistical differences between experimental groups. The Student t test was used to evaluate the difference between the averages of the groups. The results were considered statistically significant at p < 0.05.

Cytological examination
Five weeks after DMBA administration, 100% of animals developed breast cancer. The preferential localization of the tumor was in the cervicothoracic region (61.2%), regardless of the time of clinical onset of the injury. Among the animals, eight (28.5%) had multiple tumors. Tumor size ranged from 1.1 x 0.7 x 0.8 cm to 2.6 x 1.3 x 1.0 cm.
All developed tumors that were diagnosed by cytological examination were malignant. Based on the malignancy criteria described by Karim et al. (2008), the tumors showed high cellularity, anisokaryosis, anisocytosis, loose chromatin, and prominent nucleoli.

Histopathology
The neoplastic morphological types evidenced by HE staining were classified as recommended by Russo et al. (2000) in the following three classes: tubular carcinoma (16 cases), ductal (9 cases), and papillary ductal (3 cases). The qualitative analysis showed that there was no necrosis in the untreated tumor (Figure 1). In the tumors subjected to PDT, the higher the fluence, the greater was the area of necrosis.

RTq-PCR
Concerning the gene expression, the analysis of BCL-2 gene showed seven cases (25%) with higher expression: three were the group treated with 150 J / cm2 (G4), two were the group treated with 100 J / cm2 (G3), one was the group treated with 50 J / cm2 (G2), and one was the control group (G1) (RQ = 2.07 to 7.43). The sample that showed decreased expression in BCL-2 also belongs to the G4 group (4%) (RQ = 0.21). The other samples (20 cases) exhibited normal gene expression (RQ = 0.56 to 1.71). The expression level of this gene ranged from 0.56 to 7.43, with a mean of +1.87/-1.68. There was no significant statistical change in the expression of this gene among the different groups ( Figure 2A).  Figure 2C).

Discussion
The chemically-induced mammary tumors in rodents are widely used models for the study of cancer pathogenesis and molecular mechanisms involved in the triggering and progression of the neoplastic process (Russo & Russo, 1996;Vohra et al. 2021). However, the use of this model in the study of PDT is rare. There are anatomical and physiological similarities between the model used in this study and female human beings (Russo et al. 1990;Heffelfinger et al. 2000).
Mammary tumors induced by a single dose of DMBA vary morphologically from hyperplasia to adenomas and carcinomas (Terada et al. 1995;Kocdor et al. 2000;Barros et al. 2004;). In the present study, the diagnosed malignant neoplasm showed that the model was properly established.
The type of necrosis observed in tumors treated with PDT was hemorrhagic necrosis. Comparing the fluences and the depth of the breast tissue necrosis, this study revealed that the higher the fluence, the greater is the area of necrosis and vascular injury. When the photosensitizers were administered to the patient at a given concentration and received proper irradiance, a series of reactions was triggered, resulting in the formation of reactive oxygen species (ROS), inducing cell death. The amount of free radicals produced during therapy generally depends on the fluence applied to the tissue (George & Abrahamse 2016;Duanmu et al. 2011;Wyld et al. 2001).
Irradiation of bulky tumors can be achieved through optical fibers with attached cylindrical diffuse fiber tips. Depending on the volume to be treated, several fiber diffusers can be positioned simultaneously inside the tumor. One of the challenges for diffusers is to provide a homogeneous light emission, leading to variations in emission intensity, influencing the distribution of light in the tissue. It is known that this heterogeneity in the emission profile of cylindrical light diffusers interferes with the distance and depth of light distribution, which can lead to different concentrations of ROS in the tissue. In addition, the heterogeneity of tumor tissue also interferes with light distribution and, consequently, with the production of ROS, considering that there are several absorbers, spreaders in the tissue, as well as the presence of necrotic tissue. These factors can affect the effectiveness of PDT and the type of cell death (Investigation of non-uniformly emitting optical fiber diffusers on the light distribution in tissue) (Ströbl et al. 2020). Chiu et al. (2003). demonstrated that the cell lines of human prostate cancer DU -145 are resistant to PDT-induced apoptosis due to the absence of the Bax gene. Moreover, according to Srivastava et al.(2001), the treatment of RIF1 cells (mouse tumor model system) with PDT (Pc 4 -Phthalocyanines) resulted in down-regulation of the Bcl-2 gene, which could trigger the apoptotic pathway. The damage caused to Bcl-2 by PDT was also described by Xue et al. (2001). In our study, all the DMBAinduced mammary tumors expressed protein and Bcl-2 and Bax genes, which could prevent the apoptotic process. The PDTinduced cell death observed in our study resulted from necrosis, as confirmed by histopathology. Conflicting results could be explained by different cell culture, different photosensitizers, different radiation parameters, different Drug Light Interval, and time between treatments and euthanasia of the animals.
The inhibition and/or degradation of the erb-B2 protein after the completion of the PDT treatment, as demonstrated by Koval et al. (2010) and reported by Martinez-Carpio et al. (2010) apparently occurred also in this study due to the low expression of erbB2, regardless of the fluences [27]. According to , the ErbB2 receptor is related to the progression of the breast tumor and signs of malignancy, such as migration and invasion, and its overexpression is usually associated with several types of cancer. The clinical study by Zheng et al. (2019) found a clinical correlation between a higher serum concentration of the HER2 extracellular domain (ECD) and worsening in several patients. Thus, a reduction in HER-2 expression and a consequent reduction in the ErbB2 protein may be a positive indicator of PDT success (Appert-Collin et al. 2015).
Modulation of BAX, BCL2, and HER-2 gene expression was not observed between the control and the groups treated with PDT. Differences observed between the gene level and protein expression observed in our study could be explained by epigenetic abnormalities such as the modification of CpG-island methylation (Yeh et al. 2003) or post-translational modifications.
The DLI (Drug-light interval) affects the response to PDT. When a hematoporphyrin (photosensitizer) is used and a large interval is adopted, the action of PDT is cellular. However, if a smaller interval is used, the vascular response is predominant.
Thus, the PDT may be associated with the inhibition or activation of several genes involved in cell death.
Another factor is the type of photosensitizer used. According to Luo et al. (1997), the Bcl-2 family genes may be affected differently depending on the PS used. The Photogem photosensitizer used in this study is a complex mixture of monomeric and aggregated porphyrins that causes damage to the mitochondria when activated by light, also affecting gene expression (Perlin et al. 1985). Photosensitizers located in the mitochondria often cause cell death by apoptosis (Peng & Nesland 1996;Graham et al. 2003). The Phthalocyanines are macrocyclic compounds of synthetic origin whose structure is formed by units of isoindol kept together by nitrogen bridges. The PC4, a photosensitizer used in most mentioned studies, will connect to the mitochondria, endoplasmic reticulum, and the Golgi complex . The Hypericin, another photosensitizer also mentioned, is a type of antroperilene quinine photoactive pigment found in plants of the genus Hypericum. It targets mainly mitochondria, and one of its main advantages over porphyrin compounds is its low tendency to aggregate (Diwu & Lown, 1990;Halder at al. 2005).
Therefore, they represent different molecules with different degrees of aggregation that might affect the penetration of the photosensitizer in the cell. Furthermore, the concentration of the photosensitizer, its affinity for different organelles, and its distribution may also influence the response mechanisms to PDT (Teiten et al. 2003). Silva et al. (2014) suggested that the apoptosis induction is directly dependent on the duration of the tissue therapy, and that the increased expression of pro-apoptosis genes and decreased expression of anti-apoptotic genes in photodynamic therapy indicates that these genes are more directly involved with the PDT-induced apoptosis, using PDZ as a photosensitizer. These results showed the importance of these genes in activating PDT-induced apoptosis.
Another factor that affects the evaluation of the PDT response is the time of analysis of the post-PDT sample. Depending on this period, we might have observed other pathways of cell death such as necroptosis and pyroptosis. There are many doubts regarding the role of necroptosis in the development of neoplasms. There are indications that necroptosis inhibition is a strategy against metastases. Cancer cells may also show decreased necroptosis mediators, suggesting that this pathway of cell death may negatively regulate carcinogenesis (Najafov et al. 2017). Pyroptosis is a cell death pathway dependent on caspases. This pathway is activated by specific inflammatory agents, leading to the death of neoplasic cells, inhibiting their proliferation, and assisting in the prevention of metastases (Fang et al. 2020).

Conclusion
PDT does not seem to induce the modulation of genes related to apoptosis under the irradiation parameters used with Photogem and analyzed after 30h of therapy. The results of this study indicate that the type of cell death stimulated by PDT in breast tumors is necrosis under the conditions of this study. This study provides data preliminary studies on the effect of photodynamic therapy in breast cancer and should be explored in future studies.