Ian F. Tannock

Lysosomes, lysosomal enzymes, and cancer

Publisher Summary Lysosomes are membrane-enclosed cytoplasmic organelles, which posses an acidic interior that contains many hydrolytic enzymes. Their major function is in the the degradation of macromolecules, which may be cellular or foreign in origin. This chapter discusses the properties of lysosomes and their enzymes that are relevant to malignancy. Lysosomal hydrolases are synthesized and glycosylated in the rough endoplasmic reticulum. Lysosomes play an important role in the malignant process. Aspects of carcinogenesis, invasion and metastasis, and shrinkage of neoplasms in response to therapy have all been attributed to lysosomes or their products. Lysosomes can be studied by several methods, such as by ultracentrifugation isolation, enzyme activity assay, histochemical techniques, and electron microscopy. The activity of lysosomal enzymes in human tumors is compared to the normal tissue of origin. Elevation in the serum level of lysosomal enzymes is reported in both cancer patients and tumor-bearing animals. Lysosomal enzyme activity is estimated in different regions of tumors. Lysosomal enzymes are associated with invasiveness and metastatic potential in some experimental tumors. Lysosomes play a role in the uptake, processing, or efflux of some cytotoxic agents and might provide mechanisms leading to some forms of drug resistance.

Chemotherapy for adenocystic carcinoma

Seventeen patients with adenocystic carcinoma have received 34 adequate trials of chemotherapy at Princess Margaret Hospital since 1969. There have been five objective responses to chemotherapy in 4 cases, lasting from five to 24 months, and 3 other patients have had stabilized disease for a period of five to seven months. Responding and stabilized patients all had symptomatic improvement. Most responses were to 5‐fluorouracil, the preferred drug for initial treatment of metastatic disease or locoregional disease that cannot be controlled by means of surgery or radiation therapy. Cancer 46:452–454, 1980.

Third line chemotherapy in patients with metastatic breast cancer: an evaluation of quality of life and cost.

Many patients with metastatic breast cancer receive several types of chemotherapy, although it is recognized that there is a declining probability of response. A major problem confronts oncologists in deciding when to recommend to patients that no further chemotherapy should be given. To address this problem we have assessed prospectively, health‐related quality of life (HRQL) and costs of health care for 35 patients with metastatic breast cancer receiving third line chemotherapy in a representative clinical situation. HRQL and utilities were measured longitudinally using the EORTC QLQ‐C30 questionnaire and the time trade‐off method. Patients received a median of 2 cycles of chemotherapy and lived a median of 4.3 months. Twelve patients (34%) had substantial (> 10 points) improvement in the Global QL subscale and more than 30% of patients had similar changes in emotional and social function. The median baseline utility score was 0.9 and utilities correlated poorly with HRQL subscale. Eighteen patients had measurable disease and one patients experienced a partial response. Grade 3/4 toxicity occurred in 30% of patients. The average cost of management from study entry to death was CDN

Experimental Chemotherapy and Concepts Related to the Cell Cycle

17,260 (~US

The influence of expression of P-glycoprotein on the penetration of anticancer drugs through multicellular layers.

12,000). Sixteen percent of this cost was associated directly with chemotherapy while hospital admissions and outpatient visits accounted for 50% and 14% of the total cost respectively. We conclude that: (a) many patients receiving third line chemotherapy maintain or improve indices of HRQL despite short survival and a low response rate: this might be due to chemotherapy, paclebo effect, or a shift in frame of reference for HRQl; (b) patients were unwilling to trade quantity for quality of life; and (c) response rates and survival may be overestimated in patients selected for clinical trials.

Factors that influence the penetration of methotrexate through solid tissue.

Scheduling of chemotherapy is limited by damage to normal tissues, and tolerated schedules are dependent on normal tissue recovery. Most anticancer drugs are more toxic to proliferating cells and the fall and recovery of granulocyte counts after chemotherapy may be explained by the effect of drugs on rapidly proliferating precursor cells in the bone marrow. It is argued that serious toxicity due to myelosuppression most often occurs because of damage to proliferating precursors that may be recognized in bone marrow rather than to stem cells. In contrast, therapy that is aimed at producing cure or long-term remission of tumours must be directed at killing tumour stem cells. The evidence that tumours contain a limited population of cells which can repopulate the tumour after treatment (and are therefore tumour stem cells) is reviewed critically. While there is quite strong evidence for a limited population of target cells, evidence from studies on metastases suggests that the tumour cells which may express this stem cell property may change with time. The stem cell concept has major implications for predictive assays. Although colony-forming assays appear to have a sound biological background for predicting tumour response, technical problems prevent them from being used routinely in patient management. Cells in tumours are known to be heterogeneous and at least three types of heterogeneity may influence tumour response to drug treatment: the development of subclones with differing properties including drug resistance; variation in cellular properties due to differentiation during clonal expansion; and variation in properties due to nutritional status and micro-anatomy. Heterogeneity in drug distribution within solid tumours may occur because of limited drug penetration from blood vessels, and nutrient-deprived cells in solid tumours may be expected to escape the toxicity of some anticancer drugs as well as being resistant to radiation because of hypoxia. This may occur both because nutrient-deprived cells have a low rate of cell proliferation, and also because of poor drug penetration to them. There is a need for improved understanding of the mechanisms that lead to cell death in tumours. If these mechanisms were understood, it might be possible to simulate them by therapeutic manoeuvres. Recent research from our laboratory suggests that the combination of low extracellular pH and hypoxia may be very toxic to cells in nutrient-deprived regions. Drugs which limit the cells ability to survive in regions of acid pH may provide strategy for therapy of nutrient-deprived cells.

Flow cytometric calibration of intracellular pH measurements in viable cells using mixtures of weak acids and bases

The success of chemotherapy in the treatment of solid tumours may be limited by cellular mechanisms leading to drug resistance and/or by the slow penetration of drugs through tissue, resulting in a steep concentration gradient from tumour blood vessels. One mechanism leading to the development of multidrug resistance is overexpression of the membrane‐based export pump P‐glycoprotein (P‐gp). The relationship between expression of P‐gp by constituent cells and the penetration of P‐gp substrates through tissue was studied by comparing the penetration of P‐gp substrates through multicellular layers derived from either wild‐type or P‐gp overexpressing cell lines. P‐gp reversal agents were added to confirm the contribution of P‐gp in influencing the penetration of its substrates. Our data indicate: 1. penetration of the P‐gp substrates, 99mTc‐sestaMIBI and 14C‐doxorubicin, is greater through multicellular layers formed from P‐gp overexpressing cell lines as compared with wild‐type cells; 2. the addition of agents that inhibit the function of P‐gp results in decreased penetration of these substrates through multicellular layers with P‐gp expression. There was no effect of P‐gp reversal agents on penetration of 14C‐sucrose or of 3H‐5‐fluorouracil (non‐substrate controls). Our data suggest that the administration of agents that inhibit the function of P‐gp might have opposing effects on therapeutic index in solid tumours: increased sensitivity of perivascular tumour cells but decreased penetration of P‐gp substrates to more distal cells. These effects may explain, in part, the limited therapeutic benefit for solid tumours that has accrued from use of agents that reverse the effects of P‐gp. Int. J. Cancer 86:101–107, 2000.

Biology of Tumor Growth

Limited penetration of anticancer drugs through tumour tissue is an important factor that may limit therapeutic effects against solid tumours. Here we report studies of the penetration of radiolabelled methotrexate through multicellular layers (MCL) of murine EMT‐6 and human MCF‐7 cells grown on semiporous teflon membranes. The penetration of methotrexate was only about 25% of that through the teflon membrane alone after 6 hr. This was true for all methotrexate concentrations examined from 10 μM to 3 mM. The presence of folic acid at concentrations above 0.1 mM and of acidic conditions decreased the uptake of methotrexate into single cells and enhanced tissue penetration. Hypoxic conditions and the presence of folinic acid (leucovorin) had no effect on penetration or uptake of methotrexate. Our data provide evidence that tissue penetration of methotrexate is through the extracellular space, that its distribution in solid tissue may be limited and that it may be possible to improve its tissue penetration. Int. J. Cancer 91:120–125, 2001.

Failure of short-course multiple drug chemotherapy to benefit patients with recurrent or metastatic head and neck cancer

We describe a new method for calibrating intracellular pH (pH1) measurements by flow cytometry, based on the null point method proposed originally by Eisner et al. (Pflügers Arch 413:553-558, 1989). The method involves suspending cells loaded with pH-sensitive dyes, such as SNARF-1 or BCECF, in defined mixtures of the weak acid butyric acid and the weak base trimethylamine. Only the uncharged forms of these agents freely permeate the plasma membrane. The weak acid donates protons intracellularly, whereas the weak base accepts them. In accordance with the Henderson-Hasselbalch equation, when cells are exposed to these mixtures, the steady-state pHi is displaced, and the fluorescence signal reflects this new pHi. The null point method described by Eisner et al. derives pHi by determining the molar ratio of acid to base that produces no change in fluorescence signal. In this paper, we show that it is not necessary to obtain the true null point, because a calibration curve can be derived from pseudo null values whose pHi is defined by the equation pHi = pHe -0.5 log [(AT)/(BT)], where pHe is the extracellular pH, and (AT) and (BT) are the total concentrations of weak acid and base in the suspension. We refer to this as the pseudo null calibration method. It is rapid, technically simple, and reproducible. Compared with the widely used nigericin calibration method, it is not influenced by the intracellular potassium concentration; therefore, it may give a more reliable estimate of the absolute value of pHi.

Influence of cell concentration in limiting the therapeutic benefit of P-glycoprotein reversal agents

Why do malignant tumors initially grow rapidly, then more slowly? What accounts for the different rates of growth among different tumors? Can the answers to these and related questions enable us to improve the results of cancer therapy? With advanced technology, we have come a long way in penetrating the mechanisms of tumor cell kinetics, but to date clinical strategies have not kept pace.