Gemcitabine as a chemotherapeutic agent in NSCLC
Dr Snehlatha Salian
An antimetabolite, gemcitabine (GemzarR), had been approved by the US Food and Drug Aministration (FDA) in 1996 for use in first line treatment of different stages of pancreatic adenocarcinomas. The most common lung malignancy worldwide is the non-small cell lung cancer (NSCLC), which is also one of the leading causes of cancer related deaths today.
Cigarette consumption worldwide grew from a few billion to 5.5 trillion in the last 100 years. As per World Health Organization (WHO) estimates deaths due to tobacco consumption related reasons to be approximately five million annually. Lung cancer is usually diagnosed long after the patients have started or stopped smoking. Non-smokers or the passive smokers, due to the environmental factors, form 10 percent of the lung cancer population.
The cytotoxic activity of gemcitabine has been identified to be active against breast, bladder, ovarian, pancreatic, head and neck, cervical, renal, small cell lung and NSCLC cancers. Gemcitabine, a nucleoside analog, is a deoxycytidine with di-fluoro moieties at the 2′ position (Figure 1). The antiproliferative activity of this drug has been reported in number of tumours, including solid tumours. It is reported that gemcitabine was used in single drug chemotherapy in phase I and phase II clinical trials of advanced NSCLC. The object of this short review is to give an insight into the various aspects of gemcitabine as a chemotherapeutic drug in treatment of NSCLC.
Gemcitabine is transported into the plasma membrane by nucleoside transporters. There are two families of nucleoside transporters that differ on the basis of their function.
1) Equilibrative nucleoside transporters (ENTs) are facilitative and equilibrative viz, hENT1, hENT2, hENT3 and hENT4
2) Concentrative nucleoside transporters (CNTs) are sodium-dependent transporters capable of transporting the nucleoside against the concentration gradient viz, hCNT1, hCNT2 and hCNT3.
Human ENT1 and hENT2 are the two members of ENT family that are well studied and reported. They differ in their sensitivity to inhibition by nitrobenzylmercaptopurine (NBMPR). ENT1 is an NBMPR sensitive transporter while ENT2 is an NBMPR insensitive transporter. They are also capable of transporting the nucleobases. The equilibrative transporter, hENT3, was identified in 2001 and hENT4 was added to the ENT family in 2002. All the four isoforms of equilibrative nucleoside transporters are ubiquitously present at relatively varying levels in different tissues. The highest concentration of hENT2 is present in the skeletal muscle. The ENT1 and ENT2 are found in the basolateral transmembrane along with the CNTs.
The sodium-dependent hCNT1, hCNT2 and hCNT3 differ in their substrate specificities. hCNT1 transports pyrimidine nucleosides and hCNT2 transports purine nucleosides and uridine. hCNT3 transports both the pyrimidine and the purine nucleosides. hCNT1 is present in all epithelial cells and in the immune system cells. Human CNT2 and CNT3 are present in all tissues. The levels of CNTs are comparatively lesser than ENTs. Once gemcitabine enters the cell through the ENTs, it reaches equilibrium between the inside and outside of the plasma membrane. However, the CNTs present increase the concentration of the drug inside the cells by accumulation against the concentration gradient, as shown in Figure 2. Gemcitabine is transported by hENT1, hENT2 and hCNT1 into the plasma membrane but not by hCNT2.
Mechanism of gemcitabine action
Deoxycytidine nucleoside analog, gemcitabine (2’,2′-difluorodeoxycytidine, dFdC), is transported by the nucleoside transporters. Inside the plasma membrane, the rate-limiting enzyme deoxycytidine kinase (dCK, EC 220.127.116.11) brings about the phosphorylation of dFdC to its difluorodeoxycytidine monophosphate form (dFdCMP).
This is spontaneously converted to its difluorodeoxycytidine 5’diphosphate (dFdCDP) and difluorodeoxycytidine 5’triphosphate (dFdCTP) forms by the enzyme nucleoside kinases (NK). The triphosphate form of the drug is the active metabolite showing cytotoxic activity against the tumour cells and it also induces cell death by inhibiting DNA synthesis. Enzyme dCK is inhibited by dFdCTP.
Cytoplasmic 5′ nucleotidase brings about the dephosphorylation of dFdCMP. This acts in the opposite direction to that of the dCK enzymes which bring about the phosphorylation dFdC.
Deactivation of gemcitabine via deamination to cytidine is catalysed by the enzyme cytidine deaminase (CDA, EC 18.104.22.168). A part of the monophosphate form (dFdCMP) of the drug is converted to difluorodeoxyuridine monophosphate (dFdUMP), an inactive metabolite, by the enzyme dCMP deaminase (dCMPDA). The cytidine diphosphate (CDP) formed is reduced by ribonucleotide reductase (RNR) to deoxycytidine diphosphate (dCDP). It is then converted to deoxycytidine triphosphate (dCTP) by the enzyme CTP synthetase (CTPS). The dFdCTP inhibits ribonuleotide reductase, which regulates the production of nucleotide required for the DNA synthesis and repair. Hence, there is an increased incorporation of dFdCTP into DNA that blocks the DNA synthesis (termed ‘masked chain termination’). This also leads to an increase in the number of DNA single strand breaks, chromosome break and micronuclei formation in the cell. Figure 3 shows diagramatic representation of the mode of entry and the metabolism of the drug inside the plasma membrane.
The incorporation of gemcitabine into DNA in the form of dFdCTP causes cell death. Nucleoside analogs are also known to enter the RNA and induce apoptosis. Gemcitabine inhibits dCMP deaminase, and thus, regulates the catabolism of dFdCTP. The salvage pathway enzymes, namely, RNR, dCMPDA and CTPS are inhibited by dFdCTP, which regulate the production of deoxynucleotides that are required for the DNA synthesis. This indicates the ability of gemcitabine to autoactivate its mechanism of action because of its shorter half-life of elimination process of 15-20 minutes.
The process of cell death due to the treatment of drug has been worked out well in NSCLC. The 5-diphosphate and the 5-triphosphate of dFdC enter the DNA and RNA to bring about the cytotoxic effects. The cytotoxicity of gemcitabine via the dFdCTP was reported to be through the inhibition of the nucleoside metabolic enzymes like the RNR, dCMPDA and CTPS. This reduces the deoxynucleotide pool in the cell resulting in the masked chain termination. The dFdCTP is highly potent in bringing the cytotoxic affects on the tumour cell.
Role of gemcitabine
Gemcitabine regulates a number of functions in tumour cells
1) p53 gene: In H460 cells of NSCLC, gemcitabine induces apoptotic pathway by activation of caspase-8, which is a mitochondrial dependent apical caspase. It subsequently activates the down stream caspases. The cells with wild type p53 gene undergo apoptosis at a higher level than the mutated one. In NSCLC there exist two apoptotic pathways—one is p53 independent and the other is p53 dependent.
2) Radiation therapy: Gemcitabine is a potent radiosensitising agent when adminstered two hours prior to radiation. There is decrease in the dATP pool and an increase in the levels of dFdCTP. The cells are sensitised to radiation by the enzyme dCK. The action of gemcitabine in chemoradiation therapy in NSCLC shows good response rate.
3)Transcription factor NF-kB: Chemotherapy activates NF-kB in NSCLC. There is higher expression transcription factor NF-kB in A549 cells that regulate the inhibitor of apoptosis protein (IAP-1) and mRNA. The IAP-1 may play a role in modulating the sensitivity to the drug.
4) Multidrug resistance: Multidrug resistance (MDR) phenotype is characterised by an overexpression of the membrane efflux pumps P-glycoprotein (PgP) or the multidrug resistance associated protein (MRP). NSCLC cells have MDR, that causes cellular stress resulting in increased gemcitabine metabolism and sensitivity.
5) Factors affecting apoptosis: DNA dependent protein kinase and p53 gene play a role in gemcitabine treated cells. A sensor complex is formed by the DNA dependent protein kinase (DNA-PK) and p53 gene. This interacts with the gemcitabine containing DNA to trigger the subsequent signals for apoptosis.
6) Nucleotide pool: Paclitaxel, when administered prior to gemcitabine, showed higher cytotoxic effect. However, the cytotoxic effects were lower when gemcitbine was given prior to paclitaxel. The reason is higher accumulation of dFdCTP due to paclitaxel. In addition, the incorporation of gemcitabine into DNA and RNA resulted in increase in cell death by apoptosis.
Gemcitabine is also used in multiple drug therapies for treatment of advanced solid tumours. In NSCLC phase I trails for gemcitabine plus paclitaxel and gemcitabine plus vinorelbine, it was used with good efficacy. In phase II—gemcitabine and paclitaxel, and phase III clinical trials—gemcitabine and cisplatin, combined gave better results in locally advanced and metastatic NSCLC as first line of treatment compared to single drug therapy. In multiple drug therapy studies, gemcitabine with cisplatin and vinorelbine has been effective in stage III NSCLC.
The sequence of drug administration in combination therapy played an important role in enhancing the cytotoxic effect of the drug eg, paclitaxel given prior to gemcitabine showed favourable results. Paclitaxel acts by blocking cells at G2/M phase and gemcitabine arrests the cells at S phase of the cell cycle. When used in combination with drugs viz, cisplatin, etopside, mitomycin C and topotecan, gemcitabine showed synergism.
Determinants of sensitivity to gemcitabine
The role of nucleoside transpoters in transporting the therapeutic agents and the possibility of modulating them have also been reviewed. The nucleoside transporters play an important role in the gemcitabine sensitivity There are two major factors responsible for deciding the drug sensitivity—nucleoside transporters and nucleoside analog metabolic enzymes. Thus, it is possible to regulate the equilibrative and concentrative nucleoside transporters to increase the drug gemcitabine concentration inside the plasma membrane, which in turn could increase the efficacy of the drug.
Figure 1 shows Gemcitabine chemical structure Figure 2 shows the uptake of gemcitabine by nucleoside transporters
Figure 3 shows the mode of transport by nucleoside transporters, and the metabolic pathway of gemcitabine
The phosphorylation and deamination of gemcitabine inside the plasma membrane.
(The author has a PhD degree from Pune University. This review is related to her work carried out on nucleoside transporters and gemcitabine used in NSCLC at Hollings Cancer Centre, Charleston, South Caroline, USA. She can be contacted on firstname.lastname@example.org)