Are adenocarcinomas of the cardia resistant, as gastric cancers, to quinone anti-cancer drugs and radiation therapy?
R. Izutani, S. Asano, M. Imano, M. Kato, H. Ohyanagi (Osaka),
J. Chihara (Akita)
Adenocarcinoma of the distal esophagus and cardia, a common disease in some parts of the world including Japan, is a biologically aggressive disease, and an epidemiological survey has revealed that its incidence is becoming dominant among esophageal cancer tissues.
In Japan, 5-fluorouracil (5-FU), a metabolic antagonist, is most frequently used against adenocarcinoma of the cardia as the primary selection of anticancer drug. However, a mass killing of cancer cells cannot be expected by 5-FU, and thus, cytotoxic drugs such as alkylating agents and anticancer antibiotics are combined; combined chemotherapy with adriamycin (ADM), mitomycin-C (MMC), and cisplatin (CDDP) is used as a standard combination. While these regimens have led to improvement of the resection rate, they have not improved the morbidity rate [1-3]. In the present circumstances, adenocarcinoma of the cardia is thought to be resistant to radiation therapy and anticancer drugs.
In this article, we will discuss why adenocarcinoma of the cardia shows resistance to therapeutic methods from the aspects of active oxygen and antioxidants. Radiation therapies and anticancer drugs now employed for cancer treatment are in many cases closely associated with the production of active oxygen in their action mechanisms, and the relationship between therapeutic resistance and active oxygen scavengers produced by the tumor itself is drawing attention. Manganese superoxide dismutase (MnSOD), one of the active oxygen scavengers, is considered to eliminate superoxide produced in mitochondria [4, 5]. Reactive oxygen species (ROSs) are produced by inflammation, hyperoxia, and reperfusion after ischemia as well as by radiation therapy and quinone anti-cancer drugs, and the induction of MnSOD has been observed then . We examined tissue type differences in adenocarcinoma of the distal esophagus and cardia and found a greater increase in MnSOD mRNA and MnSOD protein in adenocarcinoma of the cardia than esophageal squamous cancer; the difference in radiation sensitivity could be attributable to the tissue type difference. The increases in MnSOD mRNA and protein are believed to indicate the activity of strong defense mechanisms against oxygen metabolites, and this activity is speculated to be a cause of resistance against radiation therapy and quinone anticancer drugs.
Oxygen radicals and cancer therapy
Mammalian tissues contain three main forms of SOD: Cu/ZnSOD, MnSOD and EC (extracellular) SOD. Cu/ZnSOD is a copper- and zinc containing enzyme located in the cytoplasm of various cells, whereas MnSOD is a manganese-containing enzyme present in mitochondria. Cu/ZnSOD is seldom induced and its amount of expression is scarcely changed in comparison to MnSOD. ECSOD is high molecular weight SOD present in the serum, and its expression is restricted to a number of organs including the lungs and pancreas; its degree of expression is small compared to MnSOD and Cu/ZnSOD . MnSOD is induced at the time of various oxidative stresses and is thought to regulate cellular redox balance [8, 9].
Cancer treatment methods associated with free radicals and active oxygen are largely divided, as shown in Table I. The destruction of cancer cells by radiation therapy is described as a direct action causing cleavage of the DNA strand by generating caution radicals by the action of electromagnetic waves on the DNA, and an indirect action causing cleavage of the DNA strand by generating hydroxyl radical (*OH ) by the action of radiation decomposition of body water.
Many anticancer drugs being used at present cause cleavage in the DNA strand through interaction with DNA. In other words, anticancer drugs adhere to DNA strands when they are taken into cells, and there, some of them generate OH while others interfere with DNA replication by intercalating to the DNA strand, or there is a mixed type of both actions. The production of oxygen radicals by anti-cancer drugs such as ADM, MMC, bleomycin (BLM), and CDDP have been implicated in the mechanisms of the anti-tumor effect of these drugs [10-13].
In the attempt to elucidate the biochemical mechanisms of the resistance of cancer cells to anticancer drugs, studies on the association of the redox (reduction and oxidation) system with anticancer sensitivity and resistance are particularly advanced. Our study group examined sensitivity to ADM, MMC, melphalan (L-PAM) and vincristine (VCR) in clones (cSOD) expressing MnSOD mRNA prepared by gene transduction of human MnSOD cDNA to a Chinese hamster ovary (CHO) cell strain, in comparison to control clones . Significantly higher resistance was shown by cSOD only to ADM and MMC, quinone anticancer drugs, compared to the control clone, while resistance to L-PAM and VCR did not differ between the two. In other words, cSOD showed resistance only to quinone anticancer drugs whose antitumor action is associated with the production of ROSs. Moreover, an examination of sensitivity to g-ray irradiation revealed that cSOD showed significantly higher resistance compared to the control . These findings demonstrated that a high expression of MnSOD would allow cells to be resistant to combination therapy of g-ray irradiation and quinone anticancer drugs having ROSs producibility.
Expression of MnSOD mRNA and amount of MnSOD protein
in adenocarcinoma of the cardia
We have developed a quantitative polymerase chain reaction (RT-PCR) assay to determine the amount of MnSOD mRNA in mucosal sections obtained from resected specimens from patients with esophageal cancer and adenocarcinoma of the cardia .
With use of the MnSOD mRNA quantitative PCR assay, extremely high levels of MnSOD mRNA were detected in adenocarcinoma tissue from the cardia, whereas a very low level of MnSOD mRNA was observed in non-cancerous tissue (Table II). In esophageal squamous carcinoma, the level of MnSOD mRNA was significantly increased, when compared with non-cancerous tissue (Table II). The level of MnSOD mRNA was elevated in adenocarcinoma tissue from the cardia in comparison with esophageal squamous cell carcinoma tissue (Table II).
An enzyme-linked immunosorbent assay (ELISA) was used in this study for the measurement of MnSOD contents. There were significantly higher levels of MnSOD protein in cancerous tissue compared with non-cancerous mucosa of cardia. MnSOD protein was also increased in cancerous tissue from esophageal squamous cancers compared with non-cancerous tissue (Table III). Tissue levels of MnSOD protein were significantly higher in the adenocarcinoma tissue than in the esophageal squamous carcinoma (Table III).
For immunohistological examination, surgically resected and formalin-fixed tissue samples were stained by the ABC (avidin-biotin-peroxidase complex) method. Anti-MnSOD antibody was supplied by N. Taniguchi (Department of Biochemistry, Osaka University Medical School). In esophageal carcinoma, granular positive staining was shown in the cytoplasm of the cells in the cancerous region in comparison to the healthy region (Figure 1). In adenocarcinoma of the cardia, strong diffused positive staining was shown in the cancerous region compared to the non-cancerous region (Figure 1). Staining was stronger in the adenocarcinoma of the cardia than in the esophageal carcinoma (Figure 1). The increase in intensity of immunological staining in the cytoplasm was considered to reflect an increase in MnSOD content. Our study demonstrated that MnSOD was expressed more in the cancer tissue of esophageal squamous cancer and adenocarcinoma of the cardia than in the non-cancerous tissue. A further expression of MnSOD mRNA and the amount of enzyme protein increased in the adenocarcinoma of the cardia in comparison with the esophageal squamous cancer, as did the stronger histological staining. Thus, a distinct increase in MnSOD content was observed in the adenocarcinoma of the cardia.
It has generally been accepted that esophageal squamous cancer is more sensitive than adenocarcinoma of the cardia to radiation therapy and quinone anticancer drugs; the distinct increase in MnSOD mRNA expression in adenocarcinoma of the cardia compared with that in esophageal squamous carcinoma suggests a close relation between MnSOD and the sensitivity. These increases are believed to indicate the activity of strong defense mechanisms against oxygen metabolites, and this activity is speculated to be a cause of resistance against radiation therapy and anticancer drug therapy. These findings suggested a close association between the poor therapeutic result of anticancer drugs in the adenocarcinoma of the cardia and MnSOD. While surgical resection has been considered the only standard treatment for adenocarcinoma of the cardia to date, it is difficult to acquire long-term survival by surgery alone, and clinical trials such as on the inhibition of anti-oxidant enzyme expression are necessary to achieve efficacy of anticancer drugs and radiation therapy.
Figure 1. Immunohistochemical examination of esophageal mucosa obtained from squamous cell carcinoma and adenocarcinoma. Esophageal squamous cell carcinoma showed an increase of granular positive MnSOD staining in the cytoplasm (A) (X20). The positive MnSOD staining was more prominent in the cytoplasm in adenocarcinoma than in the squamous cell carcinoma (B) (X10).
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