Sensitivity tests of tumors to cytostatic agents. I. Comparative investigations on transplanted tumors in vivo and in vitro.

With series of transplanted tumors, the activities of different cytostatic agents which directly influence the metabolism of nucleic acids (Actinomycin D, adriamycin, daunomycin, 5-fluorouracil, procarbazine, trenimon) was measured by determining 3-H-uridine incorporation in short-term (3hrs) incubations of tumor cell suspensions. Data obtained could be used to predict the response of each tumor to particular cytostatic agents in vivo. The activities of the cytostatic agents as determined using long-term tissue cultures (time of exposure of tumor cells to cytostatic agent 48 hrs were comparable to those obtained with the short-term test. In long-term cultures, determination of cell numbers gave results similar to those obtained by morphological evaluation. In SHORt-term test, differing sensitivities of tumors to cytostatics could be detected.     

Perspectives of medical therapy of solid tumours

In the course of 55 years of its existence clinical chemotherapy succeeded in curing some types of leukaemia, aggressive lymphomas and some patients with some solid tumours. Other patients with solid tumours, due to treatment with cytostatics, hormones and immunomodulators, survive longer and have a better quality of life. Further improvement of results of medical treatment of solid tumours may be foreseen from new classical cytostatics, hormones and immunomodulators and better use of known cytostatics. Liposomal forms of cytostatics are at least equally effective and less toxic than original classical cytostatics. Pegylated forms of cytostatics are more suitable for patients and and will be probably more effective than non-pegylated foms. Rational approaches to treatmet inhibiting angiogenesis and transduction of signals in tumour cells reduce the proliferation of tumour cells and achieve remission of the neoplastic disease. Inhibition of cytoplasmic tyrosine kinases and tyrosine kinases of growth factor receptors reduces also the proliferation activity of tumour cells and some clinical studies provide evidence of their effectiveness in the treatment of human tumours. Inhibitors of cycline dependent kinases stop the movement of tumour cells across some stages of the cellular cycle and thus inhibit their proliferation. Inhibitors of pharanesyl transferase prevent the activation of ras oncogenes, the formation of pharnesyl isoprenoid and its incorporation into Rh0 proteins, interfere with actin regulation, adhesion and proliferation of cells. These new drugs are less toxic than cytostatics and have a cytostatic as well as cytocidal effect. Their effectiveness is manifested by stabilization or slight regression of the tumour. To achieve an effect long-term treatment with an optimal dose is necessary which is not necessarily identical with the maximum tolerated dose and after discontinuation of treatment a relapse occurs. Combination of new inhibitors of cell division with classical cytostatics enhances the effectiveness of treatment. In the immunotherapy of tumours monoclonal antibodies are most important which have their own antitumourous activity and increase the effectiveness of cytostatics. Vaccines, similarly as gene therapy and modulators of resistance to cytostatics have so far limited indications. Rationally prepared molecules of new substances acting on new objectives of proliferation of tumour cells have a great chance to improve the results of treatment of tumours.     

Correlation between size and external temperature

Summary The reduction in size of four experimental tumours (ISIS 130 and ISIS 208 immunocytomas, S 437 mammary adenocarcinoma, S 447 colon adenocarcinoma) was investigated in LOU rats under the influence of cytostatic agents belonging to different classes (5-fluorouracil, methotrexate, vinblastine, cisplatin, doxorubicin, cyclophosphamide). External tumour and rectal temperatures were measured at the same time, twice daily, during the whole experiment. With the rectal temperature of the rats kept constant, the reduction in tumour dimensions following chemotherapy correlated via a linear relationship with the duration and degree of tumour hypothermia for the three tumours S 437, ISIS 208, ISIS 130. However, for the same reduction in tumour volume following chemotherapy, the duration and degree of transient tumour hypothermia varied according to the type of tumour and cytostatic agent studied. There was not correlation between the decrease in size of S 447 and external tumour hypothermia. Even when the reduction in tumour size was statistically significant, the hypothermic tumour phase after drug administration was not sufficient to be significant, except for vinblastine. However, the temperature of this slowly growing tumour before chemotherapy was particularly low. The measurement of the degree and duration of external tumour hypothermia of tumours following chemotherapy would represent a new physiological technique for measuring the efficacy and duration of action of cytostatic agents.     

The kinetics of cell proliferation in neuroblastomas.

The proliferation kinetics of 11histologically undifferentiated neuroblastomas were studied with an autoradiographic in vitro method (labelling with 3H-thymidine and double labelling with 3H- and 14C-thymidine). The cytokinetic parameters revealed a marked individual pattern: LI between 3.0 and 27.8%, Ts 7.2 to 17.8 hr, Tc 13.1 to 266.3 hr, Tm 0.6 to 5.1 hr, T(G1 + G2) 4.3 to 247.6 hr. The potential tumour doubling time (without cell loss) ranges from 20.4 to 415.4 hr. The growth fraction is between 0.48 and 0.58. Cytokinetic investigations of human tumours are extraordinarily important. They cannot only characterize the growth behaviour of malignant tumours, but also provide the basis for an individual therapy. The close correlation between proliferation kinetics and tumour therapy are discussed. These relations may also be summarized in a "cytokinetic therapy index", which allows the general prediction of chemosensitivity. This index is in the field of good or sufficient sensitivity in 9 neuroblastomas; in 2 cases the response to cytostatic agents is questionable from the view-point of cytokinetics.     

Übereinstimmung von Zytostatikaeffekten in vivo und im Kurzzeittest (in vitro) bei der Leukämie L 1210

Zusammenfassung Die Eignung von In-vitro-Testmethoden zur Bestimmung der Sensibilität von malignen Tumoren gegen Zytostatika hängt davon ab, wie gut Testergebnisse mit den Therapieergebnissen in vivo übereinstimmen. An der Leukämie L 1210 wurden die Zytostatikaeffekte von 3 aus verschiedenen Gruppen stammenden Zytostatika — Adriamycin, Methotrexat, Vinblastin — in vivo und in vitro gemessen und miteinander verglichen. Als Maß für den Therapieerfolg in vivo diente das Aszitesvolumen, die absolute Tumorzellzahl und der Einbau radioaktiver Nukleinsäure- bzw. Proteinsynthesevorläufer (³H-Thymidin,³H-Desoxyuridin,³H-Uridin und³H-Leucin). In vitro wurden die Zytostatikawirkungen mit einer standardisierten Methode erfaßt (3-stündige Inkubation von Zellsuspensionen mit Zytostatika; Bestimmung des Einbaues von³H-Thymidin,³H-Desoxyuridin,³H-Uridin,³H-Leucin während der 3. Stunde).Die Aszitesvolumina wurden durch alle 3 Zytostatika nicht signifikant verändert, während die Zellzahlen durch die Medikamente dosisabhängig reduziert wurden. Bei Adriamycin ist eine gute Übereinstimmung der In vitro-Ergebnisse und der in vivo gefundenen Werte des³H-Thymidin bzw.³H-Uridineinbaus sowie der Zellzahl feststellbar. Auch bei Methotrexat entsprechen sich die Ergebnisse in vivo und in vitro. Durch Vinblastin wird die Zellzahl in vivo reduziert, während die Nucleinsäure- bzw. Proteinsynthesen weder in vivo noch in vitro beeinflußt werden. Da die Zellzahl als wesentliches Kriterium eines Therapieerfolges angesehen werden muß, können Therapieerfolge durch den Kurzzeittest zwar bei Adriamycin und Methotrexat vorausgesagt werden, eine Voraussage bei Vinblastin ist aber nicht möglich.

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Correlation between the effects of cytostatic agents on leukemia L 1210 in vivo and in the short term test in vitro

Summary The usefullness of in vitro test systems for the prediction of the sensitivity or resistance of malignant tumours depends on the correlation observed between the test results and the success of therapy in vivo. The cytostatic effects of 3 agents from different groups-adriamycin, methotrexate and vinblastine-on Leukemia L 1210 cells were compared in vivo and in vitro. The success of therapy in vivo was determined from the ascites volume, absolute tumour cell count and the incorporation of radioactive nucleic acid or protein precursors (³H-thymidine,³H-deoxyuridine,³H-uridine,³H-leucine). In vitro, the effects of the cytostatics were determined using a standardised method (3-hour incubation of cell suspensions with cytostatic agents; measurement of the incorporation of³H-thymidine,³H-deoxyuridine,³H-uridine and³H-leucine during the 3rd hour).The ascites volume was not significantly altered by any of the 3 cytostatics, whereas the cell counts were reduced in a dose-dependent manner. For adriamycin, a good correlation was observed between in vivo and in vitro experiments involving the determination of³H-thymidine and³H-uridine incorporation. A good correlation was also observed between rates of biosynthesis and cell count. Comparable results were also obtained from in vivo and in vitro experiments with methotrexate. Vinblastine caused a reduction in cell count in vivo, but both nucleic acid and protein synthesis were not affected in vivo or in vitro. Since cell count must be taken as the most important criterion for success of therapy, it can be concluded that prediction of therapy results using the short term test may be possible with methotrexate and adriamycin, but not with vinblastine.

     

Augmented antitumour effects of combination therapy with TNP-470 and chemoimmunotherapy in mice

PURPOSE: To investigate antitumour efficacy of the combination of the antiangiogenic agent TNP-470 combined with chemoimmunotherapy in different tumour models in mice MATERIALS: B6D2F1 mice and BALB/c mice were inoculated in the footpad of the right hind limb with B16F10 melanoma cells or colon adenocarcinoma cells C-26, respectively. Subsequently, they received therapy consisting of TNP-470 and/or IL-12 and tumour growth was observed. In the melanoma model this therapy regimen was combined with cisplatin in a subtherapeutic dose. The antiangiogenic action of the tested agents was evaluated using tumour-induced angiogenesis assay in vivo. In order to analyse interactions between TNP-470 (or cisplatin) and IFN-gamma on tumour cells in vitro, the following methods were used: MTT assay, Western blot analysis, and flow cytometry analysis. RESULTS: Administration of the combined therapy with TNP-470 and IL-12 resulted in augmented antitumour activity in colon-26 and B16F10 melanoma models. Addition of cisplatin further enhanced efficacy of this combined therapy in the melanoma model. We showed that antitumour activity of this combined therapy is mediated by multiple mechanisms: not only is enhancement of the antiangiogenic activity mediated by TNP-470 and IL-12 but also by the synergistic cytostatic/cytotoxic action of IL-12-induced IFN-gamma and TNP-470 or cisplatin on tumour cells. The experiments revealed that TNP-470 together with IFN-gamma leads to the increased expression of p21 protein in cancer cells, which in turn may contribute to their cytostatic/cytotoxic action in vitro. CONCLUSION: Our experiments show a successful TNP-470-based combination therapy and suggest that the enhancement of the antitumour activity could be explained by a concomitant effect on both endothelial and tumour cell compartments.     

Proton pump inhibitor‐induced tumour cell death by inhibition of a detoxification mechanism

Abstract. Fais S [Istituo Superiore di Sanità (National Institute of Health), Rome, Italy]. Proton pump inhibitor‐induced tumour cell death by inhibition of a detoxification mechanism (Symposium). J Intern Med 2010; 267 :515–525. This review presents a possible new approach against cancer, as represented by inhibition of proton pumps, a mechanism used by tumour cells to avoid intracellular accumulation of toxic substances. Proton pump inhibitors (PPIs) belong to a family of pro‐drugs that are currently used in the treatment of peptic diseases needing acidity to be activated. PPIs target the acidic tumour mass, where they are metabolized, thus blocking proton traffic. Proton pump inhibition triggers a rapid cell death as a result of intracellular acidification, caspase activation and early accumulation of reactive oxygen species into tumour cells. As a whole, the devastating effect of PPIs on tumour cells suggest the triggering of a fatal cell toxification. Many human tumours, including melanoma, osteosarcoma, lymphomas and various adenocarcinomas are responsive to PPIs. This appears highly conceivable, in as much as almost all human tumours are acidic and express high levels of proton pumps. Paradoxically, metastatic tumours appear to be more responsive to PPIs being more acidic than the majority of primary tumours. However, two clinical trials test the effectiveness of PPIs in chemosensitizing melanoma and osteosarcoma patients. Indeed, tumour acidity represents a very potent mechanism of chemoresistance. A majority of cytotoxic agents, being weak bases, are quickly protonated outside and do not enter the cells, thus preventing drugs to reach specific cellular targets. Clinical data will provide the proof of concept on the use of PPIs as a new class of antitumour agent with a very low level of systemic toxicity as compared with standard chemotherapeutic agents.     

Enhanced activity of estramustine, vinblastine, etoposide, and suramin in prostate carcinoma.

Following hormonal therapy, few treatment regimens have activity in metastatic prostate cancer. Cytotoxic agents have minimal activity in this disease. However, combinations of cytotoxic agents may be beneficial. The activity of estramustine, vinblastine, etoposide, and suramin on cell growth was evaluated. Prostate specific antigen (PSA) is routinely used as a surrogate marker for disease progression. Many pharmacological agents alter PSA levels independently of their effect on tumor growth, the effect of these agents on PSA secretion was determined. Each agent was evaluated alone and in combination with the other drugs in two prostate cancer cell lines. In LNCaP cells, estramustine and suramin were cytostatic, while vinblastine and etoposide were cytotoxic. Estramustine down-regulated etoposide PSA secretion, while suramin had no effect. The effects of etoposide and vinblastine on PSA secretion were not evaluable. In PC-3 cells, only etoposide was cytotoxic. Tandem combinations were more cytotoxic than single agents in both cell lines. The addition of a third agent to the tandem combination produced less cytotoxicity. In our hands, the best combinations were estramustine/vinblastine, suramin/vinblastine, and suramin/etoposide. These combinations yielded 20-60% higher cytotoxicity than any of the drugs alone.     

The evaluation of the cyclophosphamide sensitivity of human tumours by determining the incorporation of tritiated uridine and thymidine into the nucleic acids of human tumor cells in vitro in presence of 4-hydroxycyclophosphamide (author's transl)]

A new in vitro assay for screening the sensitivity of human tumour cells against Cyclophosphamide has been developed. While biologically activated Cyclophosphamide was unsuitable because of unpurities in the material, synthetic 4-Hydroxycyclophosphamide was shown to inhibit the incorporation of tritiated uridine and thymidine into the nucleic acids of human tumour cells in vitro. 29 tumours including 14 mammarial carcinomas, 8 ovarial carcinomas and 7 other malignant tumours were tested. While 12 tumours showed a significant and 5 only a slight inhibition of the 3H-uridine incorporation in vitro. 12 tumours showed no effect. Histologically none-differentiated tumours were more sensitive against 4-Hydroxycyclophosphamide as compared with the more differentiated ones. First observations point to 4-Hydroperoxycyclophosphamide instead of 4-Hydroxycyclophosphamide as a more suitable form of activated Cyclophosphamid for the in vitro assay of Cyclophosphamide sensitiveness because of the higher stability and better availability of this compound.     

Adjuvant treatment of pancreatic carcinoma in a clinically adapted mouse resection model.

BACKGROUND: The high rate of local recurrence after radical resection of pancreatic adenocarcinoma fosters intensive efforts to develop new approaches for adjuvant treatment. The established animal models show significant limitations in simulating an adjuvant therapeutic setting. For optimal approximation to the clinical situation we therefore improved a murine orthotopic human xenotransplantation model. METHODS: Subtotal pancreatectomy in mice was performed after orthotopic inoculation of human pancreatic cancer cells and manifestation of solid tumours. The natural course of disease, tumour growth and metastases were analysed. Gemcitabine as a cytotoxic drug was tested in vitro on the cell line used in this model and the effect of adjuvant treatment with gemcitabine in vivo was investigated. RESULTS: All tumour-resected animals showed local recurrence. Organ metastases occurred in 67% in resected compared to 25% of non-resected animals. Gemcitabine in vitro was ineffective, but as adjuvant monotherapy resulted in a highly significant reduction of tumour weight and metastatic events. CONCLUSION: Subtotal pancreatectomy for xenotransplanted pancreatic cancer in SCID beige mice is feasible. Due to high rates of local recurrence and increased organ metastases, this model offers a relevant option for preclinical adjuvant testing, especially as in vitro and in vivo effects of cytotoxic drugs differ enormously.     

Influence of Prednisone and Cytostatics on Human Blood B-, T- and O-Lymphocytes in Diseases

B-, T- and O-lymphocytes detected as EAC-, E- and non-rosette forming lymphocytes were investigated in venous blood in 49 patients with connective tissue diseases, psoriasis and chronic lymphogenous leukaemia (CLL) during treatment with either prednisone alone, prednisone and cytostatic agents or cytostatic agents alone. Prednisone alone did not change the B-, T- and O-lymphocyte counts. Cytostatics alone or in combination with prednisone reduced the B- and T-lymphocyte counts concomitant with a significant increase in the O-lymphocyte count. The findings could be explained by assuming that cytostatics disturb the immunological functions of the lymphocytes and finally deprive the cells of their B- and T-markers. The optimal immunosuppressive treatment with cytostatic agents may be associated with a certain reduction of B- and T-lymphocytes which may be used as a guideline for dosage.     

A model for lymphatic metastazing after intradermal inoculation of tumour cells (author's transl)]

A model for judgement of lymphatic spread and metastasis formation is described, using intracutaneous inoculation of tumour cells (Ehrlich ascites carcinoma). The model represents an economic method for testing the effects of anticancer agents on disseminating tumour cells as well as on established tumours metastases in regional lymph nodes.     

The effect of a combination of zindoxifene and cisplatin on Dunning R3327-G prostatic carcinomas of the rat

Summary Endocrine therapy of hormone-dependent prostatic carcinomas can be very effective at the beginning. However, tumour growth eventually resumes possibly because of the presence of androgen-independent cell clones. Addition of a cytotoxic agent to the hormonally active drug at an early stage could possibly delay progression of disease. Therefore, we studied the effects of hormonal and cytostatic treatment in a rat prostate carcinoma model: freshly transplanted Dunning R3327-G prostatic tumours of the rat were treated with the partial antioestrogen zindoxifene and cisplatin alone and in combination. In addition we tested a 2-phenylindole-linked platinum complex 3-PtCl₂, which contains both effective functions. This particular complex had only a weak non-significant inhibitory effect on tumour growth. Comparing monotherapies with zindoxifene or cisplatin at various dose levels with the corresponding combinations, it became evident that the latter treatment was significantly more effective than the use of single agents. A very low dose of 0.4 mg cisplatin together with 2 mg/kg zindoxifene inhibited tumours by 91%, which was close to the effects of castration or diethylstilbestrol (1 mg). The analyses of accessory sex organs revealed much weaker oestrogenic side-effects than those observed with diethylstilbestrol at an equivalent dose. These results demonstrated that it is possible to increase the efficacy of hormonal therapy of prostatic carcinomas by conconmitant administration of cisplatin and reduce side-effects of oestrogenic drugs.Abbreviations ORG2058 16-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione - R1881 methyltrienolone - R2858 moxestrol Supported by the Deutsche KrebshilfeDedicated to Prof. Dr. W. Wiegrebe on the occasion of his 60th birthday     

PHOTODYNAMIC THERAPY OF MALIGNANT TUMOURS

Porphyrins are powerful photodynamic agents which sensitise cells so that they are damaged when exposed to light. Malignant tumours take up and retain hæmatoporphyrin to a greater extent than does normal tissue. This study is a test of the idea that hæmatoporphyrin can serve as a selective photosensitising agent to destroy tumour cells by exposure to visible light. The administration of hæmatoporphyrin followed by light therapy proved lethal to glioma cells in culture and produced massive destruction of porphyrin-containing gliomas transplanted subcutaneously in rats. Treatment with light or hæmatoporphyrin individually was without effect. Photodynamic therapy offers a new approach to the treatment of brain tumours and other neoplasms resistant to existing forms of therapy.     

Rapamycin reduces CCR5 mRNA levels in macaques: potential applications in HIV-1 prevention and treatment

G1 cytostatic drugs reduce CCR5 co-receptor expression and enhance the antiviral activity of a CCR5 antagonist in vitro. The administration of rapamycin, a G1 cytostatic agent, to three cynomolgous macaques led to decreased CCR5 messenger RNA expression in peripheral blood mononuclear cells and cervicovaginal tissue. These results support further clinical evaluation of G1 cytostatic agents such as rapamycin targeting the downregulation of CCR5 expression as a strategy for both the prevention and treatment of HIV infection.     

In Vitro Responsiveness of Human Gastric Carcinoma to Pentagastrin

The influence of pentagastrin on thymidine incorporation of five human gastric carcinomas was studied by an in vitro short-term cell suspension technique. Two of the five tumours reacted with stimulation of incorporation to the treatment. Five tumours of other aetiology did not react to similar treatment. The possibility of gastric carcinoma as a hormone-sensitive tumour is discussed.     

Correlation between in vitro and in vivo sensitivity to human leucocyte interferon in patients with multiple myeloma

26 of 32 patients with multiple myeloma (MM) were successfully tested in vitro for human leucocyte interferon (IFN) sensitivity by use of the human tumour stem cell assay (HTCA) and/or 3H-thymidine incorporation (LI). Altogether, 12 patients were sensitive to IFN in vitro and 14 were resistant. All patients received treatment with leucocyte IFN. 8 patients were classified as responders, 3 as partial responders and 15 as non-responders. Correlating the in vitro/in vivo results, we found that the in vitro tests of the myeloma cells reflected a true sensitivity in 9 patients (75%) and a true resistance in 12 patients (86%). Furthermore, stimulation by IFN was found in 9 in vitro tests, of which 8 were obtained from non-responsive patients. Our results show that in vitro testing for IFN sensitivity is of clinical importance in predicting response to IFN treatment. Also, a stimulating effect by IFN in vitro will imply an unfavourable response in vivo.     

Renal enzyme excretion in tumors and tumor therapy

None of the hitherto investigated enzymes of the urine can be used as marker for the proof of tumours. Hopeful starts in this respect are to be seen in an increased beta-glucuronidase excretion and in a decreased gamma-glutamyl transpeptidase excretion as well as in an increased LDH5/LDH1 ratio. The lysozyme excretion with the urine gives important references to the differential diagnosis, the assessment of the prognosis and therapy of acute leucoses. An importance obtained enzyme determinations in the urine for the clarification of pathobiochemical processes of the alteration of the kidney by neoplasms, tumour-lysis-syndromes, cytostatics, tumour radiation and operations of tumours. Courses of enzyme excretion during tumour therapy which as a rule show a rhythmic hyperenzymuria allow conclusion to the moment of the flow the catabolic products of the tumour at the kidney and to the different reaction of the tubular epithelium to these substances.     

Studies on the thymidine-triphosphate synthesis in malignant tumors. II. Effect of hyperthermia, Vitamin K and Cytotoxic agents (author's transl)]

Measurements of the deoxyribonucleoside triphosphate (dNTP) contents, the [14C] thymidine and deoxyuridine incorporation and the "key enzymes" of the thymidine triphosphate (dTTP) synthesis, thymidine kinase and ribonucleotide reductase, in diploid Ehrlich-ascites carcinoma, under the application of hyperthermia, vitamin K and cytocidal agents show: The effect of hyperthermia and menadion (the basic substance of the K vitamins) on the above parameters of dNTP synthesis can explain the labile effects of hyperthemia and vitamin K therapy on cancer growth. Alterations of the dNTP concentrations and demonstrable or absent inhibition of the ribonucleotide reduction with application of fluoruracil, amethopterine, cytosine arabinoside, hydroxyurea, trisethylen iminobenzochinone and daunomycin confirm and supplement our knowledge of the cytostatic action mechanism of these substances. They show moreover by the example of fluoruracil and amethopterine medication that the dTTP concentration estimation after in-vitro incubation of tumour cells with the addition of FU or methotrexat is a better measurement of the therapeutic in-vivo responsiveness of malignant tumours than the previously performed test methods.