Combating Multidrug Resistance in Cancer

Background

Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can result in death. Cancer is caused by both external factors (tobacco, chemicals, radiation, and infectious organisms) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from metabolism). Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, biological therapy, and targeted therapy.

Anyone can develop cancer. Since the risk of being diagnosed with cancer increases with the age of an individual, most cases occur in adults who are middle-aged or older. About 77% of all cancers are diagnosed in persons 55 and older.

About 5% of all cancers are strongly hereditary, in that an inherited genetic alteration confers a very high risk of developing one or more specific types of cancer. Women who have a first-degree relative (mother, sister or daughter) with a history of breast cancer have about twice the risk of developing breast cancer compared to women who do not have a family history.

However, most cancers do not result from inherited genes but from damage (mutation) that occurs during one’s lifetime. Mutations may result from internal factors such as hormones or the metabolism of nutrients within cells, or external factors such as tobacco, chemicals, and sunlight. Male smokers are about 23 times more likely to develop lung cancer than nonsmokers.

As the most frequent cancer type with high mortality rate, lung carcinomas are a major global health problem. Lung carcinomas occur equally in both sexes in developed and developing countries. This cancer type is diagnosed in 1.4 million people every year and nearly 1.2 million die. The most of lung carcinomas are detected at an advanced stage contributing to such a high mortality rate among patients.

According to SEER (Surveillance Epidemiology and End Results) the 1-year relative survival for lung cancer has slightly increased from 35% in 1975-1979 to 45% in 2005, largely due to improvements in surgical techniques and combined therapies. However, the 5-year survival rate for all stages combined is only 15%. The survival rate is 49% for cases detected when the disease is still localized, but only 16% of lung cancers are diagnosed at this early stage.

The cancer treatment during the twentieth century was based on surgery, radiation and chemotherapy. However, surgery is not curative in cases of advanced metastatic disease, and radiation and chemotherapy are limited by severe side effects and usually without ability to separate healthy from cancerous cells. Enormous genetic diversity among cancer cells is the basis of cancer resistance. Efficacy of treatment is often reduced to a subset of cells within the heterogeneous group of cancer cells. Solid tumors, the most frequent among cancers, are able to compensate the effect produced by chemotherapy and radiation. They become or are already resistant.

Multi-drug resistance (MDR) may arise due to structural and functional changes of somatic cells in the plasma membrane, within the cytoplasm, cell organelles, or nucleus. Inherent resistance is associated with the type of cells and their localization, as well as genetic changes that induce cancers.

Evolution of acquired multi-drug resistance as a consequence of chemotherapy

Under the selective pressure of drugs, a rare genetic variant of resistant cancer cells gain the advantage and multiplies: Tumor cells arise by a complex mutation and induction pathway. Cells that do not express multidrug transporters are sensitive to chemotherapy and are eliminated. In the course of chemotherapy, further mutations and selection may greatly increase the expression of multidrug transporters, which protect the tumor cells against chemotherapy.

Scheme modified from: Sarkadi B, Homolya L, Szakacs G, Varadi A. Human Multidrug Resistance ABCB and ABCG Transporters: Participation in a Chemoimmunity Defense System. Physiol Rev 2006; 86: 1179–1236

A large number of studies explore the development of resistance by using cell lines created in the laboratory, under prolonged exposure to increasing concentrations of anti-cancer drugs. Although these in vitro models do not completely correspond to the situation in vivo, their contribution to understanding the mechanisms and possibilities of reversion of resistance is important.

Economic Burden

The rapid increase in the cancer burden represents a real crisis for public health and health systems worldwide. A major issue for many countries, even among high-resource countries, will be how to find sufficient funds to treat all cancer patients effectively and provide palliative, supportive and terminal care for the large numbers of patients, and their relatives, who will be diagnosed in the coming years. According to Lance Armstrong Foundation cancers have already progressed to where they are incurable in 80 % of patients in developing countries. Evidence shows that only 5 % of global resources for cancer are spent in the developing world. Low and lower-middle income countries will make up 46 % of new cancer cases in 2009.

The costs of illness are the monetary and non-monetary losses from cancer, and economic costs are those that can be expressed in monetary units. The National Institutes of Health – National Cancer Institute estimate overall costs of cancer in USA in 2007 at $219.2 billion. Direct medical costs of $89.0 billion result from the use of resources for medical care to prevent, diagnose and treatment and for the continuing care, rehabilitation and terminal care of patients. Indirect morbidity costs of $18.2 billion come from the loss of resources – the time and productivity lost or foregone by the patient, family, friends and others from employment, volunteer activities, leisure and housekeeping. Psychosocial or indirect mortality costs of $112.0 billion come from reduced quality of life from disability, suffering and pain which force undesirable changes in lifestyle such as economic dependence, social isolation, changes or loss of job opportunities or changed conditions of living. Considering all cancers, the human capital loss is considerable lower than the value of life lost from cancer deaths ($116 billion to $232 billion vs. $961 billion, respectively, as indicated by National Cancer Institute).

The way to reduce the value of years of life lost due to cancer is to find better prevention, screening, and treatment modalities and to make sure those technologies are applied comprehensively and cost-effectively. Clearly, the value of human capital loss and life lost from cancer far exceeds the research investment (the National Cancer Institute's budget for 2008 is about $4.8 billion).

The Need

Each year 10.9 million people worldwide are diagnosed with cancer and there are 6.7 million deaths from the disease. It is estimated that there are 24.6 million people alive who have received a diagnosis of cancer in the last five years. Cancer rates could further increase by 50% to 15 million new cases in the year 2020.

According to a new World Cancer Report from the International Agency for Research on Cancer, cancer is expected to overtake heart disease as the number one killer of people around the world by the year 2010. By 2030, the number of new cancer cases is expected to rise to 27 million, with 17 million cancer deaths.

Cancer is one of the leading causes of death worldwide (12.5%) and in the EU. The European Cancer Leagues states that 2 million Europeans are diagnosed with cancer every year. In addition, the European Cancer Patient Coalition says that:

• There are more than 1.1 million cancer deaths in Europe each year;
• Every day 5,214 Europeans are diagnosed with cancer and 3,185 die from their disease;
• Lung cancer is the most common form of cancer, followed closely by colorectal cancer;
• Lung, colorectal and breast cancer account for two-fifths of the entire European population living with cancer;
• The number of Europeans with cancer will increase dramatically over the next 20 years.

According to Eurostat 2006, the age group 45-64 years (41%) is especially at risk of developing cancer. In this age group, the most common cancers in males are of the respiratory system, i.e. lung or throat cancer. Amongst women, the most common type is breast cancer, accounting for 48 deaths per 100,000 women in the EU. The highest cancer rates in the EU are found in Belgium, the Netherlands, and Luxembourg; the lowest rates are in Portugal, Greece, and Spain.

Current Treatments

Although many anticancer drugs are in research process and some of them are used in clinical practices, there is no cure for cancer yet.

The use of classical chemotherapeutics in clinical practice is empirical, based on the best-achieved response to therapy and overall survival in large clinical trails. Therapeutic regimes with two cytotoxic drugs give better result compared to the use of only one drug, but the application of combination chemotherapy is usually followed by increase in toxicity.

Classification of lung carcinomas and current chemotherapy:

Lung carcinomas	Chemotherapy
Small cell lung carcinomas (SCLC)	First-line therapy: doxorubicin, epirubicin, cyclophosphamide, iphosphamide, vincristin, vinorelbine, cisplatin, carboplatin Second-line therapy: gemcitabine, topotecan
Non-small cell lung carcinomas (NSCLC)	First-line therapy: combinations of cisplatin/carboplatin with gemcitabine, paclitaxel, docetaxel, vinorelbine, pemetrexed Second-line therapy: gemcitabine, paclitaxel, docetaxel, vinorelbine, pemetrxed New biological agents: EGFR inhibitors: cetuximab, gefitinib, erlotinib Angiogenesis inhibitors: bevacizumab, sorafenib, neovastat COX-2 inhibitors: celecoxib, rofecoxib, valdecoxib and etoricoxib

The major problem in the treatment of small cell lung carcinomas (SCLC) is the acquirement of resistance after first-line therapy, which prevents the second-line chemotherapy to extend the life of patients.

The main obstacle in non-small cell lung carcinomas (NSCLC) treatment is inherent resistance to the large scale of chemotherapeutics. Combined therapy, especially with agents that differ in their mechanisms of action, intends to increase the survival rate of NSCLC patients.

New biological agents applied independently or in combination with standard chemotherapy, provide better therapeutic options. The combinations of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) inhibitors have similar anticancer activity as a standard chemotherapy, but cause less side effects and toxicity in NSCLC patients. Selective COX-2 inhibitors can help in the prevention and treatment of NSCLC.

Our Solution

The great contribution to the understanding of multi-drug resistance (MDR) phenomenon was achieved by studying resistant cancer cell lines selected through continuous exposure to cytotoxic drugs. Sensitive line and its counterpart resistant line represent a model for investigation of the MDR mechanisms and finding effective agents to overcome resistance. High aggressiveness joint with high mitotic activity is attributed to NCI-H460, line of non-small cell lung carcinomas (NSCLC). This was the reason why we tested the development of acquired resistance to doxorubicin on NCI-H460 cell line.

Function of the multidrug/xenobiotic ABC transport pumps (transporters)

Multidrug/xenobiotic ABC transporters reside in the plasma membrane and extrude various hydrophobic and/or amphipatic xenobiotics and metabolic products. MDR1/Pgp transports hydrophobic compounds (X), while MRP1 and ABCG2 can extrude both hydrophobic drugs and intracellularly formed metabolites, e.g., glutathione or glucuronide conjugates (C-X).

Scheme modified from: Sarkadi B, Homolya L, Szakacs G, Varadi A. Human Multidrug Resistance ABCB and ABCG Transporters: Participation in a Chemoimmunity Defense System. Physiol Rev 2006; 86: 1179–1236

Resistance to doxorubicin is due to increased expression of ABC transport pumps (P-gp or MRP1) and reduced expression of targeted molecules, topoisomerase II. The identification of "cross-resistance" to structurally and functionally unrelated drugs commonly used in oncology (etoposide, paclitaxel, vinblastin and epirubicin) showed that obtained resistant cell line - NCI-H460/R developed MDR phenotype.

Although MDR phenotype obtained through selection of resistant cells in most cases does not reflect the situation in vivo, cancer cell line with high mitotic activity, such as NCI-H460, is appropriate for studying MDR mechanisms.

The acquisition of MDR phenotype through the effect of chemotherapeutic and PKC activation

DNA is the main target for the chemotherapeutics’ action. The signal from damaged DNA leads to the expression of the early response proteins (c-Jun and c-Fos) that form AP-1 complex. The binding of AP-1 to promotor region of MDR genes (mdr1, gst or topo II a) leads to their activation. All at once, protein kinase C (PKC) can induce the expression of c-Jun and c-Fos proteins and phosphorylate MDR-associated proteins (P-gp, GST or Topo IIa) leading to the promotion of MDR phenotype.

Scheme modified from:
Bredel M. Anticancer drug resistance in primary human brain tumors, Brain Research Reviews 2001; 35:161-204

The inhibition of transport proteins (P-gp or MRP1) and additional mechanisms of resistance (GSH detoxification system, altered response to apoptotic signals, activation of protein kinases, deregulation of cell cycle, changed expression of target molecules, high angiogenic potential) are required for the effective reversion of MDR.

Our research on resistant cell line (NCI-H460/R) showed that SULFINOSINE, the purine analog, inhibits several MDR mechanisms and exerts its anticancer effect alone or in combination with doxorubicin. The strategy to overcome the problem of resistance includes the combined treatment. The purine analog – SULFINOSINE can reduce the therapeutic dose of classic cytotoxic drug – doxorubicin and thus prevent its negative effect. The contribution to the treatment of cancers is more significant because, sulfinosine can inhibit the resistance caused by doxorubicin.

Sulfinosine molecule

Sulfinosine ([R,S]-2-amino-9-β-D-ribofuranosylpurine-6-sulfinamide) is the oxidized form of 6-thioguanosine. Oxidized sulfur on the sixth carbon makes sulfinosine highly reactive with great anticancer potential. Revankar et al: Synthesis and in vivo antitumor activity of 2-amino-9H-purine-6-sulfenamide, -sulfinamide, and -sulfonamide and related purine ribonucleosides. J Med Chem. 1990 Jan;33(1):121-8.

Two pathways of sulfinosine activation

• PNP = purine nucleoside phosphorilase;
• HPRT = hypoxantin phosphorybosil transferase;
• APRT = adenine phosphorybosil transferase

Sulfinosine is metabolized in its active form(s) through several metabolic pathways. Conversion into appropriate phosphorilated form involves different purine "salvage" enzymes. The existence of several metabolic activation pathways reduces the chance for acquiring resistance to sulfinosine.

Two adducts with sulfinosine

Additional contribution to the complexity of sulfinosine metabolism is its ability to form adducts with sulfhydryl compounds glutathione (GSH) and cysteine (Cys).

Advantages Over Existing Therapies

The efficacy of the new therapy modalities was shown in cell culture, on resistant NCI-H460/R cell line and has to be demonstrated in experimental orthotopic animal model, before the trials on humans.

We examined the effects of combinations of sulfinosine (SF) and doxorubicin (DOX) in sensitive and resistant human non-small cell lung carcinoma cell lines (NCI-H460 and NCI-H460/R). NCI-H460/R cells were originally selected from NCI-H460 cells and cultured in a medium containing DOX. When NCI-H460/R cells were pretreated with SF, the chemo-sensitizing effect of SF, cell cycle modulations and effect on glutathione detoxification system were examined.

Experimental data obtained on our cell culture model of resistance indicate the following beneficial effects of the proposed therapy:

• Pretreatment with SF is more potent in improving the sensitivity of NCI-H460/R cells to DOX than conventional chemo-sensitizing agent – verapamil
• The therapeutic dose of DOX is decreased
• The effects of SF on cell growth and cell cycle disturbance are prolonged
• Several MDR mechanisms are inhibited by SF

• A: sulfinosine (SF) causes dose-dependent cell growth inhibition (50-80 %) in sensitive NCI-H460 and resistant NCI-H460/R lines in appropriate therapeutic range
• B,C: Continuous treatment with doxorubicin (DOX) increased the level of mdr1 expression in NCI-H460/R line (R) 15 times compared to sensitive NCI-H460 line (P). In our experimental model, SF shows its MDR reversal potential by reducing the level of mdr1 expression for 45 % in NCI-H460/R cells (R).

Sulfinosine increases the accumulation of doxorubicin more than verapamil, the classic reversal agent

SF increases the accumulation of DOX (92 %, 117 % and 89 %) at all tested concentrations. The increase in DOX accumulation obtained with SF is significantly higher than the increase obtained with VER, which blocks P-gp pump, the molecule responsible for MDR phenotype.

The effect of sulfinosine is prolonged and increased in cancer cells that were left for recovery after treatment

The inhibition of cell growth of resistant NCI-H460/R line induced by SF treatment followed by recovery is 4 times higher in comparison with SF treatment without recovery

Sulfinosine induces the accumulation of cancer cells in S phase and this effect stable and prolonged

SF induces the arrest of NCI-H460/R cells, suggesting usefulness of this drug in combination with drugs currently used in clinical practice

Sulfinosine decreases the rGST concentration and exerts its influence on glutathione detoxification system
Curcumine (CUR) as GST inhibitor

SF significantly reduces the total content of glutathione S-transferases in NCI-H460 cells (by 62%), and in NCI-H460 / R cells (by 70%), suggesting that SF is involved in inhibition of additional MDR mecsanism, such as glutathione detoxification system.

Sulfinosine decreases GSH concentration
Graph shows the competition between GSH and L-Cys for sulfinosine

SF decreases GSH concentration in NCI-H460 cells by 58 %. This effect is antagonised by L-cysteine. The use of different metabolic pathways by SF contributes to its potential for the multiple reversal of MDR phenotype.